Treatment Of HIV-1 Infection And AIDS

ABSTRACT

Provided herein are compositions and methods for the treatment of a patient having an HIV-1 infection and/or AIDS. More specifically this invention provides treatment of an HIV-1 infection and/or AIDS using small molecule compounds, such as inhibitors for the activation and/or activity of caspase-1. Inhibitors for the activation and/or activity of caspase-1 also prevent the cell death of CD4 T-cells in a population of CD4 T-cells comprising HIV-1 infected CD4 T-cells and uninfected CD4 T-cells. In addition, caspase-1 inhibitors inhibit inflammation, and pyroptosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/556,054, filed Jul. 23, 2012, entitled “Treatment of HIV-1 Infectionand AIDS, claiming the benefit of U.S. Appl. Ser. No. 61/572,883, filedJul. 22, 2011, U.S. Appl. Ser. No. 61/511,023, filed Jul. 23, 2011, andU.S. Appl. Ser. No. 61/575,324, filed Aug. 17, 2011, each entitled“Treatment of HIV-1 Infection and AIDS,” the disclosures of which areincorporated herewith and herein by reference in their entireties forall purposes.

FIELD OF INVENTION

The present invention relates generally to the treatment of HIV-1infection and AIDS. More specifically this invention provides treatmentof HIV-1 infection and AIDS using compounds that prevent the death ofCD4 T-cells in HIV-1 infected patients. The invention also relates tocompositions and methods that prevent the death of CD4 T-cells in apopulation of CD4 T-cells comprising HIV-1 infected CD4 T-cells anduninfected CD4 T-cells.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus-Type 1 (HIV-1) is the etiologic agent thatis responsible for Acquired Immunodeficiency Syndrome (AIDS), a syndromecharacterized by depletion of CD4′ T-lymphocytes and collapse of theimmune system. HIV infection is pandemic and HIV-associated diseaseshave become a world-wide health problem. At present, the number ofpersons infected with the pathogenic virus, HIV, has exceeded 33,000,000all over the world, and about 2,500,000 persons are being newly infectedevery year.

Helper CD4 T-cells are the most important cells in maintaining thebody's powerful immunity and they are required for almost all our immuneresponses. As dramatically demonstrated in acquired immunodeficiencysyndrome (AIDS) patients, a person lacking CD4 T-cells cannot fend offeven microbes that are normally harmless. AIDS is caused by the humanimmunodeficiency virus (HIV), which infects and kills CD4 T-cells.

When the disease progresses from HIV-1 infection to full-blown AIDS, itis because the number of T-cells has dropped to dangerous levels. AIDSis heralded by a total lymphocyte count of less than 500/mm³ and adangerously low T-cell count of below 200/mm³. With the immune system sodepleted, the body becomes highly vulnerable to opportunistic diseases.As the term suggests, these are infections and other diseases that seizethe opportunity presented by a weakened defense system. They commonlyinclude herpes simplex infection and other herpes conditions such asshingles and the oral yeast infection, thrush; Kaposi's sarcoma,characterized by the dark lesions; CKV retinitis, a herpes virus thatcan bring blindness; meningitis, an infection of the spinal cord andbrain; cervical cancer; tuberculosis, and a formerly rare type ofpneumonia.

Despite extensive efforts over the past quarter century, the precisemechanism by which HIV-1 causes progressive depletion of CD4 T-cellsremains debated. Both direct and indirect cytopathic effects have beenproposed. When immortalized T-cell lines are infected withlaboratory-adapted HIV-1 strains, direct CD4 T-cell killingpredominates. Conversely, in more physiological systems, such asinfection of lymphoid tissue with primary HIV-1 isolates, the majorityof dying cells appear as uninfected “bystander” CD4 T-cells (Finkel etal., 1995, Nat Med 1:129-134; Jekle et al., 2003, J Virol 77:5846-5854).

Various mechanisms have been proposed to contribute to the death ofthese bystander CD4 T-cells including the action of host-derived factorslike tumor necrosis factor-α, Fas ligand and TRAIL (Gandhi et al., 1998,J Exp Med 187:1113-1122; Herbeuval et al., 2005, Blood 106:3524-3531),and viral factors like HIV-1 Tat, Vpr, and Nef released from infectedcells (Schindler et al., 2006, Cell 125:1055-1067; Westendorp et al.,1995, Nature 375:497-500). Considerable interest has also focused on therole of gp120 and gp41 Env protein in indirect cell death, although itis not clear whether death signaling involves gp120 binding to itschemokine receptor or gp41-mediated fusion. It is also unclear whethersuch killing is caused by HIV-1 virions or by infected cells expressingEnv.

Most studies have focused on death mechanisms acting prior to viralentry. Less is known about the fate of HIV-1-infected CD4 T-cells thatdo not express viral genes, in particular naive CD4 T-cells in tissuesthat are refractory to productive HIV infection (Glushakova et al.,1995, Nat Med 1:1320-1322; Kreisberg et al., 2006, J Exp Med203:865-870). In these cells, infection is aborted after viral entry, asreverse transcription is initiated but fails to reach completion (Kamataet al., 2009, PLoS Pathog 5, e1000342; Epub 100209 Mar. 1000320;Swiggard et al., 2004, AIDS Res Hum Retroviruses 20:285-295; Zack etal., 1990, Cell 61:213-222; Zhou et al., 2005, J Virol 79:2199-2210).

Human lymphoid aggregate cultures (HLACs) prepared from tonsillar tissueclosely replicate the conditions encountered by HIV in vivo and thusform an attractive, biologically relevant system for studying HIV-1infection (Eckstein et al., 2001, Immunity 15:671-682). Lymphoid organsare the primary sites of HIV replication and contain more than 98% ofthe body's CD4 T-cells. Moreover, events critical to HIV diseaseprogression occur in lymphoid tissues, where the network of cell-cellinteractions mediating the immune response deteriorates and ultimatelycollapses. Primary cultures of peripheral blood cells do not fully mimicthe cytokine milieu, the cellular composition of lymphoid tissue, northe functional relationships that are undoubtedly important in HIVpathogenesis. Finally, HLACs can be infected with a low number of viralparticles in the absence of artificial mitogens, allowing analysis ofHIV cytopathicity in a natural and preserved environment.

In studies described more fully herein (e.g., see, Examples), it wasdiscovered that the death of so-called uninfected “bystander” T-cellsinvolves abortive HIV-1 infection. More specifically, it was discoveredthat after viral entry, incomplete HIV-1 reverse transcriptase productsactivate a host defense program that elicits a coordinated proapoptoticand proinflammatory response involving activation of the enzymescaspase-1 and caspase-3.

Caspases are a family of at least fourteen cysteine-dependentaspartate-directed proteases that are key mediators in the signalingpathways for apoptosis and cell disassembly (Thornberry, 1998, Chem Biol5:R97-R103). These signaling pathways vary depending on cell type andstimulus, but all apoptosis pathways appear to converge at a commoneffector pathway leading to proteolysis of key proteins. Caspases areinvolved in both the effector phase of the signaling pathway and furtherupstream at its initiation. The upstream caspases involved in initiationevents become activated and in turn activate other caspases that areinvolved in the later phases of apoptosis.

Caspase-1, the first identified caspase, is also known as interleukinconverting enzyme or “ICE.” Caspase-1 exists as an inactive proenzyme,which undergoes proteolytic processing at conserved aspartic residues toproduce two subunits, large (caspase-1 p20 subunit) and small,(caspase-1 p10 subunit) that dimerize to form the active enzyme.Caspase-1 polypeptides are derived from various caspase-1 isoformprecursors. Caspase-1 converts the inactive precursor ofinterleukin-1-beta (pIL-1β) to the pro-inflammatory active form byspecific cleavage of pIL-1β between Asp-116 and Ala-117. Besidescaspase-1 there are also eleven other known human caspases, all of whichcleave specifically at aspartyl residues. They are also observed to havestringent requirements for at least four amino acid residues on theN-terminal side of the cleavage site.

The caspases have been classified into three groups depending on theamino acid sequence that is preferred or primarily recognized. The groupof caspases, which includes caspases 1, 4, 5 and 11, have been shown toprefer hydrophobic aromatic amino acids at position 4 on the N-terminalside of the cleavage site (preferred sequence Trp-Glu-His-Asp (SEQ IDNO: 1)). Another group, which includes caspases 2, 3 and 7, recognizeaspartyl residues at both positions 1 and 4 on the N-terminal side ofthe cleavage site, and preferably a sequence of Asp-Glu-X-Asp. A thirdgroup, which includes caspases 6, 8, 9 and 10, tolerate many amino acidsin the primary recognition sequence, but seem to prefer residues withbranched, aliphatic side chains such as valine and leucine at position 1(Leu/Val-Glu-X-Asp (SEQ ID NO: 2)).

The caspases have also been grouped according to their perceivedfunction. The first subfamily consists of caspases-1 (ICE), 4, 5, 11 and12. These caspases have been shown to be involved in pro-inflammatorycytokine processing and therefore play an important role ininflammation. Caspase-1, the most studied enzyme of this class,activates the IL-1β precursor by proteolytic cleavage. This enzymetherefore plays a key role in the inflammatory response. Applicants,however, are unaware of anything in the art suggesting the use ofcaspase-1 inhibitors in methods for the treatment of an HIV-1 infectionand AIDS and for use in related methods described herein.

The remaining caspases make up the second and third subfamilies. Theseenzymes are of central importance in the intracellular signalingpathways leading to apoptosis. One subfamily consists of the enzymesinvolved in initiating events in the apoptotic pathway, includingtransduction of signals from the plasma membrane. Members of thissubfamily include caspases-2, 8, 9 and 10. The other subfamily,consisting of the effector caspases 3, 6 and 7, are involved in thefinal downstream cleavage events that result in the systematic breakdownand death of the cell by apoptosis. Caspases involved in the upstreamsignal transduction activate the downstream caspases, which then disablethe DNA repair mechanisms, fragment the nuclear DNA, dismantle the cellcytoskeleton and finally fragment the cell.

Caspase-3 (also known as apopain, CPP-32, and YAMA) is responsible forproteolytic cleavage of a variety of fundamental proteins includingcytoskeletal proteins, kinases and DNA-repair enzymes. It is a criticalmediator of apoptosis in neurons. Caspase-3 exists as an inactiveproenzyme, which undergoes proteolytic processing at conserved asparticresidues to produce two subunits, large (caspase-3 p17 subunit) andsmall, (caspase-3 p12 subunit) that dimerize to form the active enzyme.Caspase-3 polypeptides are derived from various caspase-3 isoformprecursors. Inhibition of caspase-3 has shown efficacy in models, suchas stroke, traumatic brain spinal cord injury, hypoxic brain damage,cardiac ischemia and reperfusion injury. Inhibition of caspase-1 hasbeen shown to be beneficial in models of, e.g., rheumatoid arthritis,osteoarthritis, inflammatory bowel disease and asthma. However, as muchas Applicants are aware, hitherto, nothing in the art suggested the useof caspase-1 or caspase-3 inhibitors in methods for the treatment of anHIV-1 infection and AIDS and for use in related methods describedherein.

The present treatments available for HIV-1 infection and AIDS seek toblock one or more steps involved in the production of viral particlesand often are based on a combination of several drugs, a so-calledcocktail of inhibitors of reverse transcriptase and protease inhibitors.Treatment options involve administration of reverse transcriptaseinhibitors, inhibitors of viral protease, fusion, entry, or integrationinhibitors in different combinations to block multiple steps in theviral life cycle. This approach, termed highly active antiviral therapy(HAART) has greatly decreased morbidity and mortality in people infectedwith HIV (Palella et al., 1998, N Engl J Med 338(13):855-860). WhileHAART is quite effective and can reduce the virus back to undetectablelevels in patient's blood, it is not a cure for the patient, because thevirus is still present in the immune cells, and the disease can reappearat any time due to emergence of drug-resistant viruses; upondiscontinuation of therapy viremia peaks and rapid progression to AIDSis frequently observed. Furthermore, the immunodeficiency and the HIV-1specific T-cell dysfunction persist during HAART. This therapy requireslife-long treatment and the treatment is very expensive. The cost of thedrugs alone often exceeds USD 15,000. There are, in addition, severalother problems associated with this therapy; difficulties with patientcompliance (complicated drug regimens), development of resistantviruses, non-ideal pharmacokinetics and side effects such as, forexample, suppression of bone-marrow and long-term metabolic effects.

The global health crisis caused by HIV-1 is unquestioned, and whilerecent advances in drug therapies have been successful in slowing theprogression of AIDS, there is still a need to find a safer, moreefficient, less expensive way to control the virus, to treat patientshaving an HIV-1 infection or AIDS. Applicants herein provide novelmethods for the treatment of HIV-1 infection and AIDS that overcome theafore-mentioned problems.

BRIEF SUMMARY OF THE INVENTION

This application discloses the surprising finding that compounds thatinhibit the activation and/or activity of caspase-1 are useful for thetreatment of HIV-1 infection and AIDS, for slowing disease progressionin HIV-1 infected patients, for treatment of patients being infectedwith HIV-1, for treatment of patients having AIDS, for preserving CD4T-cells, for inhibition of pyroptosis, and for decreasing inflammation.

In one aspect, the invention provides a method for the treatment of apatient having an HIV-1 infection, of a patient suspected of having anHIV-1 infection, or of a patient having AIDS. In some embodiments, thismethod comprises the step of selecting a patient having an HIV-1infection, suspected of having an HIV-1 infection or having AIDS. Insome embodiments, this method comprises the step of administering to thepatient in need of such treatment a therapeutically effective amount ofa caspase-1 inhibitor.

The patient being treated according to a method of the present inventionmay have cells comprising incomplete HIV-1 nucleic acids. The incompleteHIV-1 nucleic acids may be the product of an abortive HIV-1 reversetranscription reaction.

In some embodiments, the patient being treated according to a method ofthe present invention may have developed a resistance against ananti-HIV-1 compound.

In some embodiments of the method for the treatment of a patient havingan HIV-1 infection, of a patient suspected of having an HIV-1 infection,or of a patient having AIDS, the caspase-1 inhibitor administered to thepatient in need of such treatment, is a caspase-1 inhibitor selectedfrom the group of caspase-1 inhibitors having Formula 1a, 1b, 2, 3, 4,4.1, 4.2, 4.3, 5, 6, 7, 8, 8.1, 8.2, 9, 9.1, 10, 11, 12, 13, 14, 15, 16,16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 17, 17.1, 17.2, 17.3, 17.4,17.5, 17.6, 17.7, 17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15,17.16, 17.17, 17.18, 17.19, 17.20, 17.21, 17.22, 18A, 18B, 18.1, 18.2,19, 19A, 19B, 20, 20A, 21, 21A, 22, 22A, 23(I), 23(II), 23(III), 24, 25,26, 27, a caspase-1 inhibitor depicted in FIGS. 30 and 31 andcombinations thereof.

In some embodiments of the method for the treatment of a patient havingan HIV-1 infection, of a patient suspected of having an HIV-1 infection,or of a patient having AIDS, the caspase-1 inhibitor is selected fromthe group of caspase-1 inhibitors consisting of BACMK(Boc-Asp(Obzl)-CMK, z-VAD (Z-Val-Ala-Asp), BocD, LY333531, casputin,Ac-DQMD-CHO (Ac-Asp-Met-Gln-Asp-CHO) (SEQ ID NO: 3), CV-1013, VX-740,VX-765, VX-799, Ac-YVAD-CMK (SEQ ID NO: 4), IDN-5370, IDN-6556,IDN-6734, IDN-1965, IDN-1529, z-VAD-fmk (Z-Val-Ala-Asp(OMe)-Fluoromethyl ester), z-DEVD-cmk (SEQ ID NO: 5), Z-DEVD (SEQ ID NO: 6),Ac-YVAD-fmk (SEQ ID NO: 7), z-Asp-Ch2-DCB, Ac-IETD (SEQ ID NO: 8),Ac-VDVAD (SEQ ID NO: 9), Ac-DQMD (SEQ ID NO: 10), Ac-LEHD (SEQ ID NO:11), Z-WEHD (SEQ ID NO: 12), Z-WEHD-fmk (SEQ ID NO: 13),Z-WE(OMe)HD(OMe)-fmk (SEQ ID NO: 14), Z-YVAD (SEQ ID NO: 15), Z-YVAD-fmk(SEQ ID NO: 16), Ac-YVAD-cmk (SEQ ID NO: 17), Ac-VEID (SEQ ID NO: 18)and single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof. A preferred caspase-1 inhibitor isVX-765.

In some embodiments of the method for the treatment of a patient havingan HIV-1 infection or of a patient suspected of having an HIV-1infection, or of a patient having AIDS, the caspase-1 inhibitor isselected from the group of caspase-1 inhibitors consisting ofBoc-Phg-Asp-fmk, Boc-(2-F-Phg)-Asp-fmk, Boc-(F₃-Val)-Asp-fmk,Boc-(3-F-Val)-Asp-fmk, Ac-Phg-Asp-fmk, Ac-(2-F-Phg)-Asp-fmk,Ac-(F₃-Val)-Asp-fmk, Ac-(3-F-Val)Asp-fmk, Z-Phg-Asp-fmkZ-(2-F-Phg)-Asp-fmk, Z-(F₃-Val)-Asp-fmk, Z-Chg-Asp-fmk,Z-(2-Fug)-Asp-fmk, Z-(4-F-Phg)-Asp-fmk, Z-(4-Cl-Phg)-Asp-fmk,Z-3-Thg)-Asp-fmk, Z-(2-Fua)-Asp-fmk, Z-(2-Tha)-Asp-fmk,Z-3-Fua)-Asp-fmk, Z-(3-Tha)-Asp-fmk, Z-(3-Cl-Ala)-Asp-fmk,Z-(3-F-Ala)-Asp-fmk, Z-(F₃-Ala)-Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fmk,Z-(3-C₁₋₃-F-Ala)-Asp-fmk, Z-(2-Me-Val)Asp-ink, Z-(2-Me-Ala)-Asp-fmk,Z-(2-i-Pr-β-Ala)-Asp-fmk, Z-(3-Ph-β-Ala)-Asp-fmk, Z-(3-CN-Ala)-Asp-fmk,Z-(1-Nal)-Asp-fmk, Z-Cha-Asp-fmk, Z-3-CF₃-Ala)Asp-fmk,Z-(4-CF₃-Phg)-Asp-fmk, Z-(3-Me₂N-Ala)-Asp-fmk, Z-(2-Abu)-Asp-ink,Z-Tle-Asp-fmk, Z-Cpg-Asp-fmk, Z-Cbg-Asp-fmk, Z-Thz-Asp-fmk,Z-(3-F-Val)-Asp-fmk, Z-2-Thg)Asp-fmk and single stereoisomers, mixturesof stereoisomers, pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments of the method for the treatment of a patient havingan HIV-1 infection or of a patient suspected of having an HIV-1infection, or of a patient having AIDS, the method comprises the step ofadministering to the patient an anti HIV-1 compound. In someembodiments, HAART is co-administered to the patient.

In some embodiments of the method for the treatment of a patient havingan HIV-1 infection or of a patient suspected of having an HIV-1infection, or of a patient having AIDS, the patient has a reduced T-cellcount of less than 1,000/mm³, less than 750/mm³ or less than 500/mm³.

The invention also provides a method for preventing the death of a CD4T-cell in a population of CD4 T-cells comprising HIV-1 infected anduninfected CD4 T-cells. This method can be practiced in vitro and invivo.

In some embodiments of the method for preventing the death of a CD4T-cell in a population of CD4 T-cells comprising HIV-1 infected anduninfected CD4 T-cells, the method comprises the step of contacting thepopulation of CD4 T-cells with a caspase-1 inhibitor, hereby preventingthe death of the CD4 T-cell. When practiced in vivo, the methodcomprises the step of selecting a patient in need of having the CD4T-cell contacted with a caspase-1 inhibitor.

In some embodiments of the method for preventing the death of a CD4T-cell in a population of CD4 T-cells comprising HIV-1 infected anduninfected CD4 T-cells, the caspase-1 inhibitor is selected from thegroup of caspase-1 inhibitors having Formula 1a, 1b, 2, 3, 4, 4.1, 4.2,4.3, 5, 6, 7, 8, 8.1, 8.2, 9, 9.1, 10, 11, 12, 13, 14, 15, 16, 16.1,16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 17, 17.1, 17.2, 17.3, 17.4, 17.5,17.6, 17.7, 17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15, 17.16,17.17, 17.18, 17.19, 17.20, 17.21, 17.22, 18A, 18B, 18.1, 18.2, 19, 19A,19B, 20, 20A, 21, 21A, 22, 22A, 23(I), 23(II), 23(III), 24, 25, 26, 27,28, 29, 30, 31A, 31B, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, acaspase-1 inhibitor depicted in FIGS. 30 and 31 and combinationsthereof.

In some embodiments of the method for preventing the death of a CD4T-cell in a population of CD4 T-cells comprising HIV-1 infected anduninfected CD4 T-cells, the caspase-1 inhibitor is selected from thegroup of caspase-1 inhibitors consisting of BACMK (Boc-Asp(Obz1)-CMK,z-VAD (Z-Val-Ala-Asp), BocD, LY333531, casputin, Ac-DQMD-CHO(Ac-Asp-Met-Gln-Asp-CHO) (SEQ ID NO: 3), CV-1013, VX-740, VX-765,VX-799, Ac-YVAD-CMK (SEQ ID NO: 4), IDN-5370, IDN-6556, IDN-6734,IDN-1965, IDN-1529, z-VAD-fmk (Z-Val-Ala-Asp(OMe)-Fluoro methyl ester),z-DEVD-cmk (SEQ ID NO: 5), Z-DEVD (SEQ ID NO: 6), Ac-YVAD-fmk (SEQ IDNO: 7), z-Asp-Ch2-DCB, Ac-IETD (SEQ ID NO: 8), Ac-VDVAD (SEQ ID NO: 9),Ac-DQMD (SEQ ID NO: 10), Ac-LEHD (SEQ ID NO: 11), Z-WEHD (SEQ ID NO:12), Z-WEHD-fmk (SEQ ID NO: 13), Z-WE(OMe)HD(OMe)-fmk (SEQ ID NO: 14),Z-YVAD (SEQ ID NO: 15), Z-YVAD-fmk (SEQ ID NO: 16), Ac-YVAD-cmk (SEQ IDNO: 17), Ac-VEID (SEQ ID NO: 18) and single stereoisomers, mixtures ofstereoisomers, pharmaceutically acceptable salts or prodrugs thereof. Apreferred caspase-1 inhibitor is VX-765.

In some embodiments of the method for preventing the death of a CD4T-cell in a population of CD4 T-cells comprising HIV-1 infected anduninfected CD4 T-cells, the caspase-1 inhibitor is selected from thegroup of caspase-1 inhibitors consisting of Boc-Phg-Asp-fmk,Boc-(2-F-Phg)-Asp-fmk, Boc-(F₃-Val)-Asp-fmk, Boc-(3-F-Val)-Asp-fmk,Ac-Phg-Asp-fmk, Ac-(2-F-Phg)-Asp-fmk, Ac-(F₃-Val)-Asp-fmk,Ac-(3-F-Val)Asp-fmk, Z-Phg-Asp-fmk Z-(2-F-Phg)-Asp-fmk,Z-(F₃-Val)-Asp-fmk, Z-Chg-Asp-fmk, Z-(2-Fug)-Asp-fmk,Z-(4-F-Phg)-Asp-fmk, Z-(4-Cl-Phg)-Asp-fmk, Z-3-Thg)-Asp-fmk,Z-(2-Fua)-Asp-fmk, Z-(2-Tha)-Asp-fmk, Z-3-Fua)-Asp-fmk,Z-(3-Tha)-Asp-fmk, Z-(3-Cl-Ala)-Asp-fmk, Z-(3-F-Ala)-Asp-fmk,Z-(F₃-Ala)-Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fmk, Z-(3-C₁₋₃-F-Ala)-Asp-fmk,Z-(2-Me-Val)Asp-ink, Z-(2-Me-Ala)-Asp-fmk, Z-(2-i-Pr-β-Ala)-Asp-fmk,Z-(3-Ph-β-Ala)-Asp-fmk, Z-(3-CN-Ala)-Asp-fmk, Z-(1-Nal)-Asp-fmk,Z-Cha-Asp-fmk, Z-3-CF₃-Ala)Asp-fmk, Z-(4-CF₃-Phg)-Asp-fmk,Z-(3-Me₂N-Ala)-Asp-fmk, Z-(2-Abu)-Asp-ink, Z-Tle-Asp-fmk, Z-Cpg-Asp-fmk,Z-Cbg-Asp-fmk, Z-Thz-Asp-fmk, Z-(3-F-Val)-Asp-fmk, Z-2-Thg)Asp-fmk andsingle stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof.

The invention also provides a method for inhibiting pyroptosis. Thismethod can be practiced in vitro and in vivo.

In some embodiments of the method for inhibiting pyroptosis, the methodcomprises the step of administering a caspase-1 inhibitor to a patientin need of inhibiting pyroptosis. In some embodiments, the methodcomprises the steps of selecting a patient having cells undergoingpyroptosis and administering to the patient a pharmaceutically effectiveamount of a caspase-1 inhibitor. Thereby pyroptosis is inhibited.

In some embodiments of the method for inhibiting pyroptosis, thecaspase-1 inhibitor is selected from the group of caspase-1 inhibitorshaving Formula 1a, 1b, 2, 3, 4, 4.1, 4.2, 4.3, 5, 6, 7, 8, 8.1, 8.2, 9,9.1, 10, 11, 12, 13, 14, 15, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6,16.7, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 17.10,17.11, 17.12, 17.13, 17.14, 17.15, 17.16, 17.17, 17.18, 17.19, 17.20,17.21, 17.22, 18A, 18B, 18.1, 18.2, 19, 19A, 19B, 20, 20A, 21, 21A, 22,22A, 23(I), 23(II), 23(III), 24, 25, 26, 27, 28, 29, 30, 31A, 31B, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, a caspase-1 inhibitordepicted in FIGS. 30 and 31 and combinations thereof.

In some embodiments of the method for inhibiting pyroptosis thecaspase-1 inhibitor is selected from the group of caspase-1 inhibitorsconsisting of BACMK (Boc-Asp(Obzl)-CMK, z-VAD (Z-Val-Ala-Asp), BocD,LY333531, casputin, Ac-DQMD-CHO (Ac-Asp-Met-Gln-Asp-CHO) (SEQ ID NO: 3),CV-1013, VX-740, VX-765, VX-799, Ac-YVAD-CMK (SEQ ID NO: 4), IDN-5370,IDN-6556, IDN-6734, IDN-1965, IDN-1529, z-VAD-fmk(Z-Val-Ala-Asp(OMe)-Fluoro methyl ester), z-DEVD-cmk (SEQ ID NO: 5),Z-DEVD (SEQ ID NO: 6), Ac-YVAD-fmk (SEQ ID NO: 7), z-Asp-Ch2-DCB,Ac-IETD (SEQ ID NO: 8), Ac-VDVAD (SEQ ID NO: 9), Ac-DQMD (SEQ ID NO:10), Ac-LEHD (SEQ ID NO: 11), Z-WEHD (SEQ ID NO: 12), Z-WEHD-fmk (SEQ IDNO: 13), Z-WE(OMe)HD(OMe)-fmk (SEQ ID NO: 14), Z-YVAD (SEQ ID NO: 15),Z-YVAD-fmk (SEQ ID NO: 16), Ac-YVAD-cmk (SEQ ID NO: 17), Ac-VEID (SEQ IDNO: 18) and single stereoisomers, mixtures of stereoisomers,pharmaceutically acceptable salts or prodrugs thereof. A preferredcaspase-1 inhibitor is VX-765.

In some embodiments of the method for inhibiting pyroptosis, thecaspase-1 inhibitor is selected from the group of caspase-1 inhibitorsconsisting of Boc-Phg-Asp-fmk, Boc-(2-F-Phg)-Asp-fmk,Boc-(F₃-Val)-Asp-fmk, Boc-(3-F-Val)-Asp-fmk, Ac-Phg-Asp-fmk,Ac-(2-F-Phg)-Asp-fmk, Ac-(F₃-Val)-Asp-fmk, Ac-(3-F-Val)Asp-fmk,Z-Phg-Asp-fmk Z-(2-F-Phg)-Asp-fmk, Z-(F₃-Val)-Asp-fmk, Z-Chg-Asp-fmk,Z-(2-Fug)-Asp-fmk, Z-(4-F-Phg)-Asp-fmk, Z-(4-Cl-Phg)-Asp-fmk,Z-3-Thg)-Asp-fmk, Z-(2-Fua)-Asp-fmk, Z-(2-Tha)-Asp-fmk,Z-3-Fua)-Asp-fmk, Z-(3-Tha)-Asp-fmk, Z-(3-Cl-Ala)-Asp-fmk,Z-(3-F-Ala)-Asp-fmk, Z-(F₃-Ala)-Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fmk,Z-(3-C₁₋₃-F-Ala)-Asp-fmk, Z-(2-Me-Val)Asp-ink, Z-(2-Me-Ala)-Asp-fmk,Z-(2-i-Pr-β-Ala)-Asp-fmk, Z-(3-Ph-β-Ala)-Asp-fmk, Z-(3-CN-Ala)-Asp-fmk,Z-(1-Nal)-Asp-fmk, Z-Cha-Asp-fmk, Z-3-CF₃-Ala)Asp-fmk,Z-(4-CF₃-Phg)-Asp-fmk, Z-(3-Me₂N-Ala)-Asp-fmk, Z-(2-Abu)-Asp-ink,Z-Tle-Asp-fmk, Z-Cpg-Asp-fmk, Z-Cbg-Asp-fmk, Z-Thz-Asp-fmk,Z-(3-F-Val)-Asp-fmk, Z-2-Thg)Asp-fmk and single stereoisomers, mixturesof stereoisomers, pharmaceutically acceptable salts or prodrugs thereof.

Also contemplated is the use of a caspase-1 inhibitor for thepreparation of a medicament for the treatment of HIV-1 infection and/orAIDS.

Also contemplated is the use of a caspase-1 inhibitor for thepreparation of a medicament for preventing the death of a CD4 T-cell ina population of CD4 T-cells comprising HIV-1 infected and uninfected CD4T-cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts massive depletion of CD4 T-cells in HLACs containing asmall number of productively infected cells. (A) Kinetics of spreadingviral infection versus depletion of CD4 T-cells after infection of HLACswith a replication-competent HIV reporter virus encoding GFP. CD4downregulation in GFP-positive cells likely represents the combinedaction of the HIV Nef, Vpu, and Env proteins expressed by this virus.Ratios of viable CD4 versus CD8 T-cells in HIV-infected and uninfectedcultures are also shown. Flow cytometry plots represent live-gatedcells, based on the forward-scatter versus side-scatter profile of thecomplete culture. These data are the representative results of sixindependent experiments utilizing tonsil cells from six differentdonors.

FIG. 2 depicts that CD4 T-cell depletion in HIV-1-infected HLACspredominantly involves non-productively infected cells. (A) Experimentalstrategy to assess indirect cell killing in HIV-1-infected humanlymphoid cultures. Fresh human tonsil tissue from a single donor isprocessed into HLAC, and then separated into two fractions. One fractionis challenged with HIV-1 and cultured for 6 days, allowing viral spread.On day 5, the uninfected fraction is treated with AZT (5 μM) and labeledwith CFSE (1 μM). On day 6, the infected and CFSE-labeled (i.e.,uninfected) cultures are mixed and co-cultured in the presence of AZT.Because of its site of action, AZT does not block viral output from theHIV-infected cells but prevents productive infection of CFSE-labeledcells. After 6 days of co-culturing, the number of viable CFSE-positivecells is determined by flow cytometry. (B) Flow cytometry analysis ofthe mixed HLACs. Indirect killing is determined by gating on liveCFSE-positive cells in the mixed cultures. Effector cells are eitherinfected or uninfected cells. (C) Extensive depletion ofnon-productively infected CD4 T-cells by HIV-1. CFSE-labeled cells mixedwith uninfected or infected cells were cultured in the presence of 5 μMAZT alone or together with 250 nM AMD3100. Data represent liveCFSE-positive cells 6 days after co-culture with infected or uninfectedeffector cells. The absence of productive infection in the CFSE-positivecells was confirmed by internal p24 staining and monitoring GFPexpression following infection with the NLENG1 HIV-1 reporter virus (notshown). (D) Preferential depletion of non-productively infected CD4T-cells by HIV-1. The absolute numbers of viable CFSE-positive CD4 andCD8 T-cells and B cells were determined. Percentages are normalized tothe number of viable CFSE-positive cells co-cultured with uninfectedcells in the presence of AZT, as depicted by (*). Error bars representstandard deviations of three samples from the same donor. Thisexperiment is the representative of more than 10 independent experimentswith more than 10 donors of tonsillar tissues. See also FIG. 3.

FIG. 3 depicts that CD4 T-cells are sufficient to induce indirectkilling in HLAC. (A) CD4 T-cells, CD8 T-cells, and B cells were isolatedfrom HLACs by positive selection on CD4, CD8 and CD19 microbeads(Miltenyi), respectively. The isolated cells were cultured incombinations and proportions that corresponded to their authenticdistribution in the complete, undivided HLAC (shown), and were infectedwith NL4-3 (“Effector Cells”). Effector cells from the complete HLACwere used as a positive control. After 5 days, uninfected complete HLACand isolated CD4 T-cells were treated with AZT and labeled with CFSE(“CFSE(+) cells”). On day 6, the indicated effectors and theCFSE-labeled cells were co-cultured in the presence of 5 μM AZT ortogether with 250 nM AMD3100. (B) After 6 days of co-culture, the numberof viable CSFE-positive CD4 T-cells was determined by flow cytometry.Percentages are normalized to the number of viable CFSE-positive CD4T-cells co-cultured with uninfected effectors in the presence of AZT.This experiment is the representative of three independent experimentsperformed with cells from three different donors.

FIG. 4 depicts that HIV-1 fusion is necessary to induce killing ofnon-productively infected cells. (A and C) Concentrations of T20 thatblock viral infection. HLACs were infected with the indicated clones ofHIV-1 in the presence of the indicated concentrations of T20 or nodrugs. One hour before incubation with the virus, cells were pretreatedwith T20 or left untreated. At 12 hours, cells were washed extensivelyand cultured under the same conditions. On day 9, the viralconcentration was determined using a p24gag FLAQ assay. The amount ofp24gag accumulated in the absence of drugs by each viral clone (A) or bySKY (C) was defined as 100%. (B and D) Effect of T20 on indirectkilling. CFSE-labeled cells were co-cultured with cells infected withthe indicated viral clones in the presence of 5 μM AZT and the indicatedconcentrations of T20. After 6 days, indirect killing in the mixedcultures was assessed. The number of viable CFSE-positive CD4 T-cellsco-cultured with uninfected cells in the presence of AZT was defined as100% (not shown). To allow successful initial infection the GIA-SKYmutants were pseudotyped with the VSV-G envelope. NL4-3, WT lab-adaptedvirus; WEAU 16-8, primary virus; SIM, T20-resistant virus; GIA-SKY,T20-dependent virus; GIA and SKY, single-domain mutant viruses.Representative data from three independent experiments with differentdonors are shown. See also FIG. 5, below.

FIG. 5 depicts extensive indirect depletion of CD4 T-cells by a primaryHIV-1 isolate. (A) Changes in plasma viral load and CD4 T-cells aftersymptom onset in patient WEAU, who was positive for HIV-1 p24gag antigenand had early clinical manifestations of acute retroviral syndrome.Arrow indicates when the 16-8 envelope was isolated, and used to derivethe WEAU 16-8 subclone. (B) CFSE-labeled cells were co-cultured withcells infected with the primary WEAU 18-6 (R5/X4) viral isolate or thelaboratory-adapted viral clone NL4-3 (X4) in the presence of 5 μM AZT ortogether with 250 nM AMD3100. After 6 days of co-culturing, the numberof viable CSFE-positive cells was determined by flow cytometry.Percentages are normalized to the number of viable CFSE-positive cellsco-cultured with uninfected cells in the presence of AZT, as depicted by(*). Error bars represent standard deviations obtained for threereplicates performed with cells from the same donor.

FIG. 6 depicts that killing of non-productively infected CD4 T-cellsrequires fusion of virions from nearby HIV-1-producing cells. (A)Supernatants from HIV-infected HLACs are less efficient at inducingindirect killing than mixing of HIV-infected and uninfected HLACs. (B)HIV-1 virions released into the medium do not participate in indirectkilling. Replacing the mixed culture with fresh RPMI every 24 hours didnot impair indirect killing. Challenging HLACs with supernatantscontaining 20-fold more histoculture-derived virions (1 μg p24/ml) thannormally accumulated in mixed cultures containing infected cells (50 ngp24/ml) did not induce indirect killing. Percentages are normalized tothe number of viable CFSE-positive cells depicted by (*). (C)CFSE-labeled cells are not killed when HIV-infected HLAC is physicallyseparated by a 1 μm-pore transwell system that allows free diffusion ofHIV-1 particles. Values represent the levels of viable CFSE-positivecells after 6 days of culture in the presence of the indicated drugs.Red, HIV-infected cells; blue, uninfected cells; green, CFSE-labeledcells. (D) Mature and immature viruses carry equivalent amounts ofenvelope protein and Blam-Vpr, but differ in their content of capsid andGag precursor. NL4-3 and TR712 viruses were generated in 293T-cells withor without amprenavir, lysed and subjected to SDS-PAGE immunoblottinganalysis for gp120, p55 Gag, p24 CA, Blam-Vpr, and free Blam. (E)Immature viruses have reduced capacity to enter cells. SupT1 cells weremock infected or infected with mature or immature NL4-3 or TR712 virionscontaining Blam-Vpr. After loading of cells with CCF2 dye, fusion wasanalyzed by flow cytometry. Percentages are the fraction of cellsdisplaying increased cleaved CCF2 fluorescence, indicating virionfusion. (F) Protease inhibitors inhibit indirect killing. CFSE-labeledcells were co-cultured with NL4-3-infected or uninfected cells in thepresence of AZT (5 μM) alone or together with AMD3100 (250 nM). To theindicated cultures were added 5 μM of Amprenavir, Saquinavir, orIndinavir. Percentages are normalized to the number of viableCFSE-positive cells depicted by error bars represent the SD obtainedwith three independent samples from the same donor. See also FIG. 7,below.

FIG. 7 depicts that spinoculation efficiently recapitulates indirect CD4T-cell killing (A, B) and that protease inhibitors do not block syncytiaformation. (A) The spinoculation procedure. (B) Flow cytometry of viableCD4 versus CD8-T-cells in HLAC three days after spinoculation in thepresence of the indicated drugs. Data represent live-gated cells, basedon the forward-scatter versus side-scatter profile of the completeculture. Of note, apoptotic cells were included in the analysis ofcaspases activity and cytokine expression (FIG. 10E, F). Drugsconcentrations: AZT 5 μM; Efavirenz 100 nM; AMD3100 250 nM; T20 10μg/ml. The data are representative results of six independentexperiments with six different donors. Note that HIV-spinoculated CD4T-cells were readily killed in the presence of AZT but not efavirenz,T20 or AMD3100. (C) Highly permissive SupT1 CD4 T leukemic cells wereinfected with NL4-3 and cultivated for 3 days to allow substantialinfection but not syncytia formation. After 3 days, cells were treatedwith 5 μM AZT, 250 nM AMD3100, 10 μg/ml T20, 5 μM amprenavir, 5 μMsaquinavir, or 5 μM indinavir or were left untreated. After 2.5 days ofadditional culture, the extent of syncytia formation in each sample wasevaluated by light microscopy. Note that protease inhibitors inhibitedindirect killing as efficiently as AMD3100 and T20, but did not alterthe function of Env proteins expressed on the surface of infected cells(i.e., allow syncytia formation), indicating that the killing signal isnot delivered directly through the infected cells. Results arerepresentative of three independent experiments.

FIG. 8 depicts that death of abortively infected CD4 T-cells is due toimpaired reverse transcription. (A) Status of mixed HLACs containingeither resting or activated CFSE-labeled cells, 4 days afterco-culturing with effector cells. Activated CFSE-labeled cells werestimulated with PHA and IL-2 48 hours before mixing, but not duringco-culturing with effector cells. To avoid direct killing of activatedCFSE-labeled cells in cultures with no drugs, cell killing wasterminated and analyzed 4 days after co-culturing. (B) AZT rendersactivated CFSE-labeled CD4 T-cells sensitive to indirect killing.Resting or activated CFSE-labeled cells were co-cultured with effectorcells in the presence of no drugs, AZT (5 μM) alone, or AZT+AMD3100 (250nM). Data are from two independent experiments performed with tonsilcells from two different donors. (C) AZT-induced killing is lost whenAZT-resistant viruses are tested. Resting or activated CFSE-labeledcells were co-cultured with cells infected with NL4-3 or HIV-1 clones#629 and #964 in the presence of no drugs, AZT (0.5 μM) alone, orAZT+AMD3100 (250 nM). AZT-sensitive and AZT-resistant sub-clones aredepicted. Data are representative of three independent experiments withthree different donors. (D) NNRTIs prevent killing of abortive infectedCD4 T-cells. Resting or activated CFSE-labeled cells were co-culturedwith infected or uninfected effector cells, in the presence of no drugs,AZT (5 μM), AMD3100 (250 nM), the NNRTIs Efavirenz (100 nM), andNevirapine (1 μM), or the integration inhibitors Raltegravir (30 μM) and118-D-24 (60 μM). Killing of resting CFSE-labeled CD4 T-cells wasblocked with equal efficiency by NNRTIs and AMD3100 (columns 15, 16),but not by integration inhibitors (columns 17, 18). In combination,NNRTIs prevented cell death induced by AZT in activated CFSE-labeledcells (compare column 38 to 44 and 45). Data are representative of fourindependent experiments with four different donors. The absolute numbersof CFSE-labeled CD8 T-cells and B cells was unaltered in theseexperiments (data not shown). Percentages are normalized to the numberof viable CFSE-positive cells depicted by (*). See also FIG. 9, below.

FIG. 9 depicts concentrations of AZT that block viral infection (A, B),demonstrates that indirect killing occurs after strong-stop DNAsynthesis (C, D), and shows that HIV-1 clones encoding defective reversetranscription do not kill HLAC CD4 T-cells (E, F). HLACs were infectedwith viruses derived from the indicated HIV proviral clones in thepresence of the indicated concentrations of AZT or in the absence ofdrugs. Six days after infection the amount of p24^(gag) in the mediumwas determined by FLAQ assay. The amount of p24^(gag) in the mediumafter infection wash (day 0) was defined as 0%. (A) In NL4-3-infectedcells the maximal inhibitory concentration was 0.5 μM AZT. (B) In cellsinfected with HIV-1 clones #629 and #964 (Larder et al., 1989, Science243:1731-1734), concentration of 0.5 μM AZT achieved a maximalinhibitory effect in the AZT-sensitive version of these clones, and halfmaximal inhibitory concentration effect in the AZT-resistant version ofthese clones. (C) Δvif NL4-3 virions incorporate 100-fold moreendogenous APOBEC3G (A3G) than WT virions. Viral incorporation ofendogenous A3G was measured by immunoblot analysis of purified NL4-3 andΔvif NL4-3 virions harvested from 1 ml of supernatant from infectedHLACs. Doses reflect 1:10 dilutions. Virions were originally generatedfrom 293T-cells that do not express APOBEC3G; therefore, physiologicallevels of endogenous A3G packaged into newly synthesized virions areshown. p24^(gag) CA levels indicate equivalent virion production byNL4-3 and Δvif NL4-3 in infected HLACs. (D) Killing of abortivelyinfected CD4 T-cells requires elongation of viral DNA. Resting oractivated CFSE-labeled cells were co-cultured with cells infected withWT or Δvif NL4-3 in the presence or absence of the following drugs, AZT(5 μM), AMD3100 (250 nM), the NNRTIs Efavirenz (100 nM), and Nevirapine(1 μM), or the integration inhibitors Raltegravir (30 μM) and 118-D-24(60 μM). Data are representative of four independent experimentsperformed with cells from four different donors. (E) A method to assessindirect killing in HLAC with non-infectious HIV-1 clones. Fresh humantonsil is processed into HLAC and cells are cultured in suspension. Atthe same time 293T-cells are transfected with 1 μg HIV-1 DNA in a24-well plate. After 12 hours, 293T-cells are washed and overlayed with3×10⁶ HLAC cells in RPMI in the presence of the indicated drugs.Virus-producing 293T-cells directly interact with target overlaying HLACcells. After 24 hours, the HLAC suspensions are collected from wells andanalyzed by flow cytometry. (F) Indirect killing of HLAC CD4 T-cells byvirus-producing 293T-cells. HLAC were cultured with the indicated drugs3 hours before co-culture with 293T-cells. Percentages are normalized tothe number of viable CD4 T-cells in HLAC overlaying non-transfected293T-cells in the presence of AZT, as depicted by (*). Error barsrepresent standard error of the mean of three experiments performedusing cells from three different HLAC donors.

FIG. 10 depicts that cytoplasmic HIV-1 DNA triggers proapoptotic andproinflammatory responses in abortively infected CD4 T-cells. (A)Critical reactions in HIV-1 reverse transcription as detected by probesmonitoring different regions within the Strong stop, Nef, and Env DNAfragments. RDDP, RNA-dependent DNA polymerase. Adapted from S. J. Flintet al., Principles Of Virology, 2000 ASM Press, Washington D.C., withpermission. (B) NNRTIs prevent accumulation of DNA elongation products.The amount of viral DNA detected by a particular probe was calculated asa fold change relative to cells treated with no drugs (i.e. calibrator).A β-actin probe was used as an internal reference. Mean cycle threshold(Ct) values of calibrator samples are depicted. CD4 T-cells wereinfected with WT NL4-3 produced in 293T-cells, or with a Δvif NL4-3collected from supernatants of infected HLAC, as described in FIG. 9C.Data are representative of two independent experiments performed withcells from two different donors. (C and D) Abortive HIV-1 infectiongenerates a coordinated proapoptotic and proinflammatory responseinvolving caspase-3 and caspase-1 activation. HLACs were spinoculatedwith no virus or with NL4-3 and AZT (5 μM), Efavirenz (100 nM), and T20(10 μg/ml), as indicated (see FIG. 7A-B). After 3 days, cells wereassessed by flow cytometry for intracellular levels of proinflammatorycytokines, serine 37 phosporylated p53, and activated caspases asindicated. Ethidium monoazide was used to exclude dead and necroticcells from the annexin V binding analysis. Data are representative ofthree independent experiments with three different donors. (E) Death ofabortively infected CD4 T-cells requires caspase activation.CSFE-labeled cells were co-cultured with effector cells in the presenceof 20 μM of Z-VAD-FMK, a general caspase inhibitor, or Z-FA-FMK, anegative control for caspase inhibitors. AZT (5 μM); AMD3100 (250 nM).Percentages are normalized to the number of viable CFSE-positive cellsdepicted by (*). Error bars represent standard error of the mean ofthree experiments from three different HLAC donors. (F) Abortive HIVinfection promotes the maturation and secretion of IL-1β in tonsillarCD4 T-cells. Isolated tonsillar CD4 T-cells were either untreated, orstimulated with PMA (Phorbol-12-myristate-12-acetate, 0.5 μM) and thepotassium ionophore nigericin (10 μM), or spinoculated with or withoutNL4-3 in the presence of AZT (5 μM), AMD3100 (250 nM), and efavirenz(100 nM) as indicated. After 3 days, half of the cells were lysed andsubjected to SDS-PAGE immunoblotting analysis. On day 5, thesupernatants from the rest of the cells were collected and subjected toSDS-PAGE immunoblotting analysis. The IL-1β antibody detects thepro-IL-1β (37 kD) and the mature cleaved form (17 kD). Data are therepresentative results of five independent experiments using tonsillarCD4 T-cells isolated from five different donors. (G) DNA reversetranscription intermediates induce an IFN-stimulatory antiviral innateimmune response (ISD). ISRE-GFP reporters were transfected with 1 μg ofHIV-1 reverse transcription intermediate products as indicated bynumbers (detailed description in FIG. 14E), empty DNA plasmid, orpolyinosinic:polycytidylic acid [poly(I:C)], and were analyzed by flowcytometry after 48 hours. Data are representative of three independentexperiments; error bars show the SD for three independent samples fromthe same experiment. See also FIG. 11 and FIG. 13.

FIG. 11 depicts critical reactions in reverse transcription and theireffect on abortive infection-mediated killing (A) and extensivedepletion of abortively infected CD4 T-cells in human spleen (B, C). (A)(Step 1) Minus-strand DNA synthesis is initiated near the 5′ end of thepregenomic mRNA from the 3′OH of the tRNA bound to the primer bidingsite (PBS.) This initial step occurs in the natural microenvironments ofHIV-1 virions before infection, and was termed natural endogenousreverse transcription (nERT) (Zhang et al., 1996, J Virol 70:2809-2824).(Steps 2-3) DNA synthesis proceeds to the 5′ end of the mRNA genome,while RNase H digests the RNA portion of the newly formed RNA-DNAhybrid, freeing the resulting short, single-stranded DNA fragment knownas the minus-strand strong-stop DNA. In RNase H-defective HIV-1 (E478Q)the viral RNA is not degraded, and reverse transcription is paused afterstrong-stop DNA synthesis (Smith et al., 1999, J Virol 73:6573-6581). Invif-deficient HIV-1 (Δvif-HIV-1) cellular A3G is packaged into thevirion and inhibits accumulation of strong-stop DNA products (Bishop etal., 2008, PLoS Pathog 4:e1000231). Interrupting reverse transcriptionat these steps prevents indirect killing, suggesting that strong-stopDNA products are not sufficient to induce a cytopathic response in CD4T-cells. (Steps 4-5) The exposed minus-strand strong-stop DNA istransferred to the 3′ end of the genome, where it hybridizes with the rregion at the 3′ end of the same or the second RNA genome, a reactionknown as first template exchange, allowing the continuation ofRNA-dependent DNA polymerization (RDDP) of the minus DNA stand. NNRTIsbind a small hydrophobic pocket near the RT active site, inducing achange in the structure of RT that blocks early RNA-dependent andDNA-dependent polymerase activities. Inhibition of DNA synthesis byNNRTIs prevents indirect killing, indicating that this step is key forinitiating the cytopathic response.

Minus-strand DNA synthesis is accompanied by partial degradation of theRNA in the resulting RNA-DNA hybrid by RNase H, exposing viral ssDNAintermediates in the host cytoplasm. Subsequently, fragments of RNA thatwere not removed by the RNase H at polypurine tract (ppt) sites, serveas primers for plus-strand DNA synthesis. Plus-strand DNA is formedbefore minus-strand DNA synthesis is completed, exposing “islands” ofviral dsDNA intermediates in the host cytoplasm. DNA chain elongationdepends on nucleotide supply and therefore occurs after uncoating, inthe host cell cytoplasm. In non-permissive cells such as most CD4T-cells in lymphoid tissues, elongation of DNA polymerization isinhibited (i.e. “Killing Zone”) (Kamata et al., 2009, PLos Pathog5:e1000342; Pierson et al., 2002, J Virol 76:8518-8531; Swiggard et al.,2004, AIDS Res Hum Retroviruses 20:285-295; Zack et al., 1990, Cell61:213-222; Zhou et al., 2005, J Virol 79:2199-2210). In turn, viralssDNA and dsDNA intermediates accumulating in the host cytoplasm aredetected by an as-yet unidentified sensor(s), and elicit a multifacetedantiviral response involving apoptotic cell death and secretion ofproinflammatory cytokines such as IL-1β. Because AZT and endogenousarrest of reverse transcript elongation occur at the same phase of virallife cycle (Arts and Wainberg, 1994, Antimicrob Agents Chemother38:1008-1016), AZT is not required to elicit the abortiveinfection-associated cell death in the nonpermissive nave CD4 T-cellspresent in HLACs. Reverse transcription scheme is adapted from S. J.Flint et al., Principles Of Virology, 2000 ASM Press, Washington D.C.,with permission.

(B and C) Fresh human spleen from a single donor were processed intoHLAC and assessed in indirect cell killing assay as described in FIG.2A. Effector spleen HLAC were stimulated with PHA and IL-2 48 hoursbefore mixing, but not during co-culturing with CFSE-labeled cells.Non-activated resting CFSE-labeled spleen cells were co-cultured withNL4-3-infected or uninfected effector cells in the presence or absenceof the following drugs, AZT (5 μM), AMD3100 (250 nM), Efavirenz (100nM), or Raltegravir (30 μM), as indicated. Graft versus host toxicitywas not observed during co-culture of spleen and tonsils even thosethese organs were obtained from different donors. Percentages werenormalized to the number of viable CFSE-positive spleen CD4 T-cellsco-cultured with uninfected effectors in the presence of AZT, asdepicted by (*). Error bars represent standard error of the mean forthree experiments performed with three different spleen and tonsildonors.

FIG. 12 depicts consequences of inhibiting early steps of HIV-1infection on CD4 T-cell death. (A) The nonpermissive state of most CD4T-cells in lymphoid tissue leads to endogenous termination of reversetranscription during DNA chain elongation (i.e. “killing zone”). As aresult, DNA intermediates accumulate in the cytoplasm and elicit amultifaceted proapoptotic and proinflammatory innate immune defenseprogram, coordinated by IFN-stimulatory DNA (ISD) response, Caspase-3,Caspase-1, and IL-1β, to restrict viral spread. Different classes ofantiretroviral drugs act at different stages of the HIV life cycle.NNRTIs like efavirenz and nevirapine inhibit early steps of DNAsynthesis and therefore prevent such response and the subsequent CD4T-cell death. AZT is less efficient at blocking DNA synthesis andtherefore unable to abrogate this response. (B) In permissive CD4T-cells reverse transcription proceeds, allowing HIV-1 to bypass the“killing zone” and move on to productive (or latent) infection.Interrupting reverse transcription by AZT traps the virus in the“killing zone” and induces cell death. EFV, Efavirenz; NVP, Nevirapine,RTGR, Raltegravir. See also FIG. 11.

FIG. 13, including FIGS. 13A, 13B, 13C, 13D, 13E, and 13E, depicts thatdrugs that inhibit ATP-sensitive potassium channel P2X7 receptor orcaspase-1 activation and/or activity prevent CD4 T-cell depletion andinflammation in HIV-infected human lymphoid tissues.

FIGS. 13A and 13B depict that cell death is caused by suicidal innateimmune responses against this viral DNA leading to caspase-1 andcaspase-3 activation.

FIGS. 13C and 13D depict that caspase-1 activation leads to inflammasomeassembly, cleavage of the pro-interleukin-10 to bioactive IL-1β and to ahighly inflammatory form of cell death called pyroptosis.

FIG. 13E depicts that healthy lymphoid CD4 T-cells but not CD8 T-cellsnor B cells present in tonsil and spleen are primed for inflammation asevidenced by constitutive expression of high levels of pro-IL-1β.

FIG. 13F depicts that caspase-1 inhibitors Z-WEHD (SEQ ID NO: 12) andZ-YVAD (SEQ ID NO: 15) inhibit inflammation and CD4 T-cell death inHIV-infected human lymphoid cultures.

FIG. 14 depicts that death of abortively infected CD4 T-cells requiresactivation and/or activity of caspases. (A) CSFE-labeled cells werepreincubated with 20 μM of Z-VAD-FMK, a general caspase inhibitor;Z-DEVD-FMK (SEQ ID NO: 18), a caspase-3 inhibitor; or Z-FA-FMK, anegative control for caspase inhibitors. After 2 hours, CFSE-labeledcells were co-cultured with NL4-3-infected or uninfected effector cellsin the presence of 5 μM AZT or together with 250 nM AMD3100, asindicated. After 4 days of co-culture the number of viable CSFE-positiveCD4 T-cells was determined by flow cytometry. Percentages are normalizedto the number of viable CFSE-positive CD4 T-cells co-cultured withuninfected effectors in the presence of AZT, as depicted by (*). Errorbars represent standard error of the mean of three experiments fromthree different HLAC donors. (B and C) HLACs were spinoculated alone orwith NL4-3 in the presence of AZT (5 μM), Efavirenz (100 nM), and T20(10 μg/ml), as indicated (spinoculation is described in detail in FIG. 7A, B). After 3 days, cells were assessed by flow cytometry for annexin Vbinding, and intracellular levels of activated caspases (Panel B), andfor cytokine expression including TNF-α, IFN-β IL-1β and serine-37phosphorylated forms of p53 indicative of DNA damage (Panel C). Afterspinoculation with NL4-3, HLAC CD4 T-cells displayed increasedintracellular levels of activated caspase-1, caspase-3, IFN-β and IL-1β,together with the apoptotic marker annexin V. Efavirenz and T20 but notAZT prevented these responses, indicating they resulted from abortiveHIV-1 infection. In contrast to CD4 T-cells, these intracellular markersin CD8 T and B cells were not altered following NL4-3 infection,indicating a selective CD4 T-cell response, and excluding non-specifictoxicity of HIV-1. Ethidium monoazide staining was used to exclude deadand necrotic cells in annexin V binding analysis. Data arerepresentative of three independent spinoculation experiments performedwith cells from three different donors. (D) depicts representativefluorescence images of ISRE-GFP reporter H35 cell line. Depicted H35cells were uninfected or infected with VSVg-pseudotyped NL4-3 in thepresence of AZT (5 μM), Efavirenz (100 nM), and Raltegravir (30 μM) asindicated. (E) depicts a schematic illustration of the synthetic reversetranscription intermediates used in FIG. 10G. Uncapped or capped HIV-1mRNA was produced by in vitro transcription. To generate the strong-stopand RNA-DNA heteroduplex reverse transcription intermediate (−) DNAprimers were annealed to the uncapped or capped HIV-1 mRNA in theindicated sites. To generate the 150-, 500-, 1500-, and 3300 bp dsDNAreverse transcription products (step 5), PCR products corresponding tothe indicated HIV-1 pregenomic mRNA were generated using specificprimers. To generate ssDNA, the PCR products were heated at 95° C. for5-10 minutes followed by 10 minutes on ice. The detailed protocol andsequences are provided in the Examples.

FIG. 15 depicts that inhibition of caspase-1 is sufficient to preventdepletion of HIV-1 CD4 T-cells in human lymphoid tissues. Pan-caspaseinhibitor Z-VAD, caspase-1 inhibitor Z-WEHD (“1”) (SEQ ID NO: 12),evafirenz (EFV), AND AMD3100 (AMD), but not caspase-6 inhibitor Z-VEID(“6”) (SEQ ID NO: 19) or control compound Z-FA, inhibit the death ofinfected CD4 T-cells. Caspase-3 inhibitor Z-DEVD (“3”) (SEQ ID NO: 6)also inhibits death of infected CD4 T cells, however, not as efficientas caspase-1 inhibitors. Data shown represent 4 different donors (SEM).

FIG. 16 depicts that caspase-1 inhibitors efficiently inhibit CD4 T-celldeath in HIV-1-infected human lymphoid tissues. (A). CD4 T-cell death inHIV-1-infected cultures was prevented by the pan-caspase inhibitor Z-VAD(“Pan-Caspase”) and by the caspase-1 inhibitor (Z-WEHD, “Caspase 1”)(SEQ ID NO: 12) as efficient as by efavirenz and AMD3100, but not by theZ-FA-FM (commercial negative control; “Control”) and caspase-6inhibitor. Treatment with caspase-3 inhibitor (Z-DEVD; “Caspase 3”) (SEQID NO: 6) prevented the death of only 50% CD4 T-cell population. Errorbars represent standard error of the mean of three experiments fromthree different HLAC donors. (B). CD4 T-cell death in HIV-infectedcultures was prevented by the caspase-1 inhibitor (Caspase-II inhibitor,Calbiochem; “Caspase 1”) as efficient as by efavirenz and AMD3100. Inthese experiments, treatment with caspase-3 inhibitor (Z-DEVD; “Caspase3”) (SEQ ID NO: 6) did not prevent the death of HIV-infected CD4 Tcells. Error bars represent standard error of the mean of threeexperiments from three different HLAC donors. (C). LDH was not releasedform infected cells treated with efavirenz. AMD3100, or caspase-1inhibitor (Caspase-II inhibitor, Calbiochem; “Caspase 1”). Treatmentwith caspase-3 inhibitor (Z-DEVD; “Caspase 3”) (SEQ ID NO: 6) did notprevent the LDH release from HIV-1 infected cells. Error bars representstandard error of the mean of three experiments from three differentHLAC donors. Details are described in Example 23.

FIG. 17 schematically depicts cellular changes occurring duringapoptosis and pyroptosis leading to cell death. Apoptosis is an active,programmed process of autonomous cellular dismantling that avoidseliciting inflammation. Pyroptosis, a pathway of cell death mediated bythe activation and/or activity of caspase-1 includes caspase-1 cleavageof the inflammatory cytokines IL-1β and IL-18 to their bioactive forms.Pyroptotic cells are not removed by phagocytosis but undergo cell lysisand release of inflammatory cellular contents. Adapted from Fink andCookson, 2005, Infection and Immunity.

FIG. 18 depicts that AIDS is associated with a progressive loss of CD4T-cells. Details are described in the Examples.

FIG. 19 depicts an ex vivo human lymphoid culture system as a model tostudy cell death in HIV-1 infection. Details are described in theExamples

FIG. 20 depicts an HIV-1 reporter system used herein. Insertion of anIRES in the Nef gene supports GFP expression and preserves wild-typelevels of Nef. A fully replication-competent virus is produced. TheHIV-1 reporter system allows simultaneous quantification of the dynamicsof HIV-1 infection and T-cell depletion. Details are described in theExamples.

FIG. 21 depicts massive depletion of CD4 T-cells after infection oftonsillar tissue with HIV-1. Details are described in the Examples.

FIG. 22 depicts an experimental strategy to explore indirect cellkilling in HIV-1-infected human lymphoid cultures. Details are describedin the Examples.

FIG. 23 depicts that soluble factors in tonsil render CD4 T-cellssensitive to indirect killing. Details are described in the Examples.

FIG. 24A depicts an experimental design to address whether HIV-1gp41-mediated fusion is necessary for depletion of non-productivelyinfected CD4 T-cells. FIG. 24B depicts that CXCR4 signaling is notsufficient to elicit indirect T-cell killing and that HIV-1gp41-mediated fusion is necessary for depletion of non-productivelyinfected CD4 T-cells. Details are described in the Examples.

FIG. 25A depicts two possible death-induced mechanisms that wereexamined herein based on the findings that indirect killing of CD4T-cells requires both gp41-mediated fusion and close interaction withHIV-1-infected cells. “A” examines whether virions are the killing unitsand if cell death is caused by fusion of HIV-1 virions to nearby CD4T-cells. “B” examines whether productively infected CD4 T-cells are thekilling units and if cell death is caused by engagement ofcell-associated Env on HIV-1-infected cells with neighboring CD4T-cells. FIG. 25B depicts the conclusion of the experimental datadescribed herein that the killing signal is delivered by virions not byproductively infected cells. Details are described in the Examples.

FIG. 26 depicts possible mechanism of HIV-1-induced indirect CD4 T-celldeath in human lymphoid tissue that were examined herein, including (1)gp120-mediated signaling through the CXCR4 receptor, (2) gp41-mediatedfusion of virus-cell or cell-cell (syncytia), (3) impaired reversetranscription, and (4) integration. Details are described in theExamples.

FIG. 27 depicts experimental findings and set-up to address whetherHIV-1 signals for maturation and release of bioactive IL-1β inabortively infected CD4 T-cells. Details are described in the Examples.

FIGS. 28 and 29 depict that HIV-1 infection causes repeating cycles ofcell death and inflammation. The surprising and unexpected findingsdescribed in the Examples leading to this conclusion include that (i)dying bystander CD4 T-cells are abortively infected with HIV-1, (ii)cell death is not caused by HIV-1 products but rather by an innateimmune response to the cytosolic viral DNA that accumulates in restingCD4 T-cells, (iii) the death of CD4 T-cells is associated with intenseinflammation (pyroptosis), (iv) tissue-based CD4 T-cells are primed forinflammation-increased pro-IL-1β, (v) resting CD4 T-cells from lymphoidtissue die following HIV-1 infection in the absence or presence of AZT,and (vi) activated CD4 T-cells are killed by HIV-1 when AZT is present.

FIGS. 30A and 30B depict compounds useful for practicing the presentinvention, in particular, methods of the present invention using acaspase-1 inhibitor. Details of those compounds and compositions aredescribed by Stierle et al. (Stierle et al., J Nat Prod (2012)75:344-350; Stierle et al., J Nat Prod (2012) 75:262-266).

FIGS. 31A, 31B, and 31C depict compounds useful for practicing thepresent invention, in particular, methods of the present invention usinga caspase-1 inhibitor. Details of those compounds and compositions weredescribed at the April 2001 American Chemical Society (ACS) meeting inSan Diego, Calif., USA.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Throughout the present specification and the accompanying claims thewords “comprise” and “include” and variations thereof such as“comprises”, “comprising”, “includes” and “including” are to beinterpreted inclusively. That is, these words are intended to convey thepossible inclusion of other elements or integers not specificallyrecited, where the context allows. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the invention.

The terms “a” and “an” and “the” and similar referents used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. Ranges may be expressed herein as from“about” (or “approximate”) one particular value, and/or to “about” (or“approximate”) another particular value. When such a range is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about” or “approximate” itwill be understood that the particular value forms another embodiment.It will be further understood that the endpoints of each of the rangesare significant both in relation to the other endpoint, andindependently of the other endpoint. It is also understood that thereare a number of values disclosed herein, and that each value is alsoherein disclosed as “about” that particular value in addition to thevalue itself. For example, if the value “10” is disclosed, then “about10” is also disclosed. It is also understood that when a value isdisclosed that is “less than or equal to the value” or “greater than orequal to the value” possible ranges between these values are alsodisclosed, as appropriately understood by the skilled artisan. Forexample, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e. g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

The headings used herein are for organizational purposes only and arenot meant to be used to limit the scope of the description or theclaims, which can be had by reference to the specification as a whole.Accordingly, the terms defined immediately below are more fully definedby reference to the specification in its entirety.

Illustrations are for the purpose of describing a preferred embodimentof the invention and are not intended to limit the invention thereto.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

As used herein, the term “about” refers to a range of values of plus orminus 10% of a specified value. For example, the phrase “about 200”includes plus or minus 10% of 200, or from 180 to 220, unless clearlycontradicted by context.

As used herein, the term “administering” means the actual physicalintroduction of a composition into or onto (as appropriate) a host orcell. Any and all methods of introducing the composition into the hostor cell are contemplated according to the invention; the method is notdependent on any particular means of introduction and is not to be soconstrued. Means of introduction are well-known to those skilled in theart, and also are exemplified herein.

As used herein, the term “administration in combination,” “combinationtherapy” or similar grammatical equivalents refers to both simultaneousand sequential administration of compounds. One or more caspase-1antagonist can be delivered or administered at the same site or adifferent site and can be administered at the same time or after adelay, preferably not exceeding 48 hours. Concurrent or combinedadministration, as used herein, means that one or more caspase-1antagonists are administered to a subject either (a) simultaneously, or(b) at different times during the course of a common treatment schedule.In the latter case, the compounds are administered sufficiently close intime to achieve the intended effect. Concurrent or combinedadministration, as used herein, also means that one or more caspase-1antagonists can be administered in combination with another compounduseful for the treatment of HIV-1 infection and AIDS.

As used herein, an “agent” or “compound” can be any chemical compound,for example, a macromolecule or a small molecule disclosed herein. Theagent can have a formula weight of less than about 10,000 grams permole, less than 5,000 grams per mole, less than 1,000 grams per mole, orless than about 500 grams per mole. The agent can be naturally occurring(e.g., a herb or a nature product), synthetic, or both. Examples ofmacromolecules are proteins, protein complexes, and glycoproteins,nucleic acids, e.g., DNA, RNA and PNA (peptide nucleic acid). Examplesof small molecules are peptides, peptidomimetics (e.g., peptoids), aminoacids, amino acid analogs, polynucleotides, polynucleotide analogs,nucleotides, nucleotide analogs, organic or inorganic compounds e.g.,heteroorganic or organometallic compounds. An agent can be the onlysubstance used by the method described herein. Alternatively, acollection of agents can be used either consecutively or concurrently bythe methods described herein.

As used herein, the term “aliphatic” means straight chained, branched orcyclic C₁-C₁₂ hydrocarbons which are completely saturated or whichcontain one or more units of unsaturation. For example, suitablealiphatic groups include substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl, alkynyl groups and hybrids thereof such as(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

As used herein, the term “alkenyl” refers to a straight or branchedchain unsaturated hydrocarbyl moiety having one or more double bonds.Examples of alkenyl groups include vinyl, allyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl and 3-(1,4-pentadienyl).The term “lower alkenyl” refers to alkenyl groups having from 2 to 4carbon atoms.

As used herein, the term “alkenylene” refers to a divalent group derivedfrom an alkenyl group and includes, for example, ethenylene, —CH═CH—,propenylene, —CH═C═CH—, and the like.

As used herein, the term “alkoxy” refers to —OR^(d) wherein R^(d) isalkyl as defined herein. Representative examples of alkoxy groupsinclude methoxy, ethoxy, butoxy, trifluoromethoxy, and the like.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent, means, unless otherwise stated, a straight (i.e.unbranched) or branched chain, or combination thereof, which may befully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include, but are not limited to, groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “lower alkyl”refers to alkyl groups having from 1 to 4 carbon atoms.

As used herein, the term “alkylene” by itself or as part of anothersubstituent means a divalent radical derived from an alkyl, asexemplified, but not limited, by CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms.

As used herein, the term “alkynyl” refers to an unsaturated alkyl groupone having one or more triple bonds. Examples of alkynyl groups includeethynyl (acetylenyl), 1-propynyl, 1- and 2-butynyl, and the higherhomologs and isomers.

As used herein, the terms “alklysulfonyl” and “haloalkylsulfonyl” referto sulfonyl (—SO₂—) moieties substituted by an alkyl or haloalkyl group,respectively.

As used herein, the term “alkylthio” refer to sulfur atoms substitutedby alkyl groups of the indicated number of carbon atoms.

As used herein, the terms “altering the immune response” or “regulatingthe immune response” or grammatical equivalents thereof, refer to anyalteration in any cell type involved in the immune response. Thedefinition is meant to include an increase or decrease in the number ofcells, such as CD4 T-cells, an increase or decrease in the activity ofthe cells, such as CD4 T-cells, or any other changes that can occurwithin the immune system. The cells may be, but are not limited to, Tlymphocytes, B lymphocytes, natural killer (NK) cells, macrophages,eosinophils, mast cells, dendritic cells, or neutrophils. The definitionencompasses both a stimulation or enhancement of the immune system todevelop a sufficiently potent response to a caspase-1 antagonist asdescribed herein, as well as a suppression of the immune system to avoida destructive response to a desirable target. In the case of stimulationof the immune system, the definition includes future protection againstsubsequent HIV-1 infection.

As used herein, the term “amount sufficient”, an “effective amount” or“therapeutically effective amount” or grammatical equivalents is thatamount of a given compound to ameliorate, or in some manner, reduce asymptom or stop or reverse progression of a disease, disorder, orcondition. In some embodiments, the desired activity of interest isdiminishing, abolishing or interfering with the physiological action ofa caspase-1 polypeptide, which provides either a subjective relief of asymptom(s) or an objectively identifiable improvement as noted by aclinician or other qualified observer. In some embodiments, the desiredactivity of interest is decreasing the amount or activity ofbiologically active IL-1β. The dosing range varies with the compoundused, the route of administration and the potency of the particularcompound. Amelioration of a symptom(s) of a particular condition byadministration of a pharmaceutical composition described herein refersto any lessening, whether permanent or temporary, lasting or transientthat can be associated with the administration of the pharmaceuticalcomposition. An “effective amount” can be administered in vivo and invitro.

As used herein, the terms “antagonist” or “inhibitor” (usedinterchangeably herein) mean a chemical substance that diminishes,abolishes or interferes with the physiological action of a polypeptide.The antagonist may be, for example, a chemical antagonist, apharmacokinetic antagonist, a non-competitive antagonist, or aphysiological antagonist, such as a biomolecule, e.g., a polypeptide, apeptide antagonist or a non-peptide antagonist. A preferred antagonistdiminishes, abolishes or interferes with the physiological action of acaspase-1 polypeptide. As used herein, a “cytokine antagonist is acompound that inhibits or blocks the expression and/or activity of acytokine, e.g. an interleukin (IL), such as IL-1β, or interferon oranother cytokine.

As used herein, the term “aryl” means, unless otherwise stated, apolyunsaturated, aromatic, hydrocarbon substituent which can be a singlering or multiple rings (preferably from 1 to 3 rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from one to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed herein. “Substituted heteroaryl” refers to a unsubstitutedheteroaryl group as defined above in which one or more of the ringmembers is bonded to a non-hydrogen atom such as described above withrespect to substituted alkyl groups and substituted aryl groups.Representative substituents include straight and branched chain alkylgroups —CH₃, —C₂H₅, —CH₂OH, —OH, —OCH₃, —OC₂H₅, —OCF₃, —OC(═O)CH₃,—OC(═O)NH₂, —OC(═O)N(CH₃)₂, —CN, —NO₂, —C(═O)CH₃, —CO₂H, —CO₂CH₃,—CONH₂, —NH₂, —N(CH₃)₂, —NHSO₂CH₃, —NHCOCH₃, —NHC(═O)OCH₃, —NHSO₂CH₃,—SO₂CH₃, —SO₂NH₂ and halo. “Arylene” and “heteroarylene” refers to adivalent radical derived from a aryl and heteroaryl, respectively.

As used herein, the term “arylalkyl” is meant to include those radicalsin which an aryl group is attached to an alkyl group (e.g., benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (e.g., a methylene group) has been replaced by, forexample, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,3-(1-naphthyloxyl)propyl, and the like.

As used herein, the term “biaryl” (when used as a group or as part of agroup) refers to a group containing the specified number of atoms andcontaining two aromatic rings which have two atoms in common. Examplesof biaryl as used herein include, but are not limited to naphthyl. Saidbiaryl groups may be optionally substituted—where not otherwisespecified, the substitutions may be one or more groups selected fromC₁-C₃alkyl, C₁-C₃alkoxy, —C(O)Me, CO₂H, CO₂Me and ═O.

As used herein, the term “biologically active” when referring to anagent or compound is art-recognized and refers to a form of an agent orcompound that allows for it, or a portion of the amount of agent orcompound administered, to be absorbed by, incorporated into, orotherwise physiologically available to a subject or patient to whom itis administered.

As used herein, the terms “caspase antagonist”, “caspase inhibitor” or“inhibitor of caspase activity” refer to a compound that is capable ofpreventing, whether fully or partially, activity of a caspasepolypeptide, as measured by any suitable assay, such as those describedand referenced herein. Preferred caspases are caspase-1 and caspase-3.Most preferred is a caspase-1.

As used herein, the term “caspase-1” refers to nucleic acids,polypeptides and polymorphic variants, alleles, mutants, andinterspecies homologues thereof and as further described herein, that:(1) have an amino acid sequence that has greater than about 85%, 90%,preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateramino acid sequence identity, preferably over a region of at least about25, 50, 75, 100, 150, 200, 250, 300, 350, or more amino acids, to asequence as deposited under GenBank Accession Nos., e.g., NP_(—)150634,NP_(—)001214, AAT72297, or NP_(—)150635; (2) bind to antibodies, e.g.,monoclonal and/or polyclonal antibodies, raised against an immunogencomprising an amino acid sequence as deposited under GenBank AccessionNos., e.g., NP_(—)150634, NP_(—)001214, AAT72297, or NP_(—)150635, orconservatively modified variants thereof or a fragment thereof; (3)modulate at least partially, indirectly or directly the production ofbioactive IL-1β; (4) specifically hybridize under stringenthybridization conditions to a nucleic acid sequence as deposited underGenBank Accession Nos., e.g., NM_(—)033292, NM_(—)001223, AY660536, orNM_(—)033293, or conservatively modified variants thereof; (5) have anucleic acid sequence that has greater than about 90%, preferablygreater than about 96%, 97%, 98%, 99%, or higher nucleotide sequenceidentity, preferably over a region of at least about 30, 50, 100, 200,500, 1000, 1,200 or more nucleotides, to a nucleic acid sequences asdeposited under GenBank Accession Nos., e.g., NM_(—)033292,NM_(—)001223, AY660536, or NM_(—)033293; (6) have at least 25, often 50,75, 100, 150, 200, 250, 300, 350, or more contiguous amino acid residuesof a polypeptide the sequence of which is deposited under GenBankAccession Nos., e.g., NP_(—)150634, NP_(—)001214, AAT72297, orNP_(—)150635; and/or (7) have at least 25, often 50, 75, 100, 150, 200,250, 300, 350, 400, 500, 600, 700, 800, 900, 1,000, 1,200, or morecontiguous nucleotides of a nucleic acid sequences as deposited underGenBank Accession Nos., e.g., NM_(—)033292, NM_(—)001223, AY660536, orNM_(—)033293. Preferred is a mammalian caspase-1. A preferred mammaliancaspase-1 is a human caspase-1. Also preferred is a simian caspase-1.

As used herein, the terms “caspase-1 antagonist” or “caspase-1inhibitor” or “inhibitor of caspase-1 activity” refer to a compound thatis capable of preventing, whether fully or partially, activation and/oractivity of a caspase-1 polypeptide, as measured by any suitable assaysuch as those described and referenced herein. A “peptide caspase-1inhibitor” comprises at least one natural amino acid or at least onenon-natural amino acid.

As used herein, the term “caspase-3” refers to nucleic acids,polypeptides and polymorphic variants, alleles, mutants, andinterspecies homologues thereof and as further described herein, that:(1) have an amino acid sequence that has greater than about 85%, 90%,preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateramino acid sequence identity, preferably over a region of at least about25, 50, 75, 100, 150, 200, 250, or more amino acids, to a sequence asdeposited under GenBank Accession Nos., e.g., NP_(—)004337,NP_(—)116786, or CAC88866; (2) bind to antibodies, e.g., polyclonaland/or monoclonal antibodies, raised against an immunogen comprising anamino acid sequence as deposited under GenBank Accession Nos. e.g.,NP_(—)004337, NP_(—)116786, or CAC88866, or conservatively modifiedvariants thereof or a fragment thereof; (3) modulate at least partially,indirectly or directly the activation of caspases 6, 7, and 9 orcleavage of amyloid-beta 4A precursor protein; (4) specificallyhybridize under stringent hybridization conditions to a nucleic acidsequence as deposited under GenBank Accession Nos., e.g., NM_(—)004346,NM_(—)032991, or AJ413269, or conservatively modified variants thereof;(5) have a nucleic acid sequence that has greater than about 90%,preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotidesequence identity, preferably over a region of at least about 30, 50,100, 200, 500, 1000, 1,500 or more nucleotides, to a nucleic acidsequences as deposited under GenBank Accession Nos., e.g., NM_(—)004346,NM_(—)032991, or AJ413269; (6) have at least 25, often 50, 75, 100, 150,200, 250, or more contiguous amino acid residues of a polypeptide thesequence of which is deposited under GenBank Accession Nos. e.g.,NP_(—)004337, NP_(—)116786, or CAC88866; and/or (7) have at least 25,often 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800,900, 1,000, 1,200, 1,500, or more contiguous nucleotides of a nucleicacid sequences as deposited under GenBank Accession Nos., e.g.,NM_(—)004346, NM_(—)032991, or AJ413269. Preferred is a mammaliancaspase-3. A preferred mammalian caspase-3 is a human caspase-3. Alsopreferred is a simian caspase-3.

As used herein, the terms “caspase-3 antagonist” or “caspase-3inhibitor” or “inhibitor of caspase-3 activity” refer to a compound thatis capable of preventing, whether fully or partially, activation and/oractivity of a caspase-3 polypeptide, as measured by any suitable assaysuch as those described and referenced herein. Some caspase-1antagonists described herein also diminish, abolish or interfere withthe physiological action of a caspase-3 polypeptide.

Throughout the present specification and the accompanying claims thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other additives, elements, components, integers or stepsnot specifically recited, where the context allows.

By “contacting” is meant an instance of exposure of at least onesubstance to another substance. For example, contacting can includecontacting a substance, such as a cell or a polypeptide to an agentdescribed herein. A cell can be contacted with the agent, for example,by adding the agent to the culture medium (by continuous infusion, bybolus delivery, or by changing the medium to a medium that contains theagent) or by adding the agent to the extracellular fluid in vivo (bylocal delivery, systemic delivery, intravenous injection, bolusdelivery, or continuous infusion). The duration of contact with a cellor group of cells is determined by the time the agent is present atphysiologically effective (biologically active) levels or at presumedphysiologically effective (biologically active) levels in the medium orextracellular fluid bathing the cell. In the present invention, forexample, a virally infected cell (e. g. an HIV-1 infected cell) or acell at risk for viral infection (e. g., before, at about the same time,or shortly after HIV-1 infection of the cell) is contacted with anagent. The term “contacting” is used herein interchangeably with thefollowing: combined with, added to, mixed with, passed over, incubatedwith, flowed over, place in direct physical association with anothersubstance, etc.

As used herein, the terms “cycloalkyl” and “heterocycloalkyl”, bythemselves or in combination with other terms, represent, unlessotherwise stated, cyclic versions of “alkyl” and “heteroalkyl”,respectively. The term “cycloalkyl” refers to a saturated cyclichydrocarbon having 3 to 8 carbon atoms, and 1 to 3 rings that can befused or linked covalently. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Cycloalkyl groups useful in thepresent invention include, but are not limited to, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl, and the like. Bicycloalkylgroups useful in the present invention include, but are not limited to,[3.3.0]bicyclooctanyl, [2.2.2]bicyclooctanyl, [4.3.0]bicyclononane,[4.4.0]bicyclodecane (decalin), spiro[3.4]octanyl, spiro[2.5]octanyl,and so forth. Examples of heterocycloalkyl include, but are not limitedto, 1 (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and“heterocycloalkylene” refer to a divalent radical derived fromcycloalkyl and heterocycloalkyl, respectively.

As used herein, the term “cycloalkenyl” refers to an unsaturated cyclichydrocarbon having 3 to 15 carbons, and 1 to 3 rings that can be fusedor linked covalently. Cycloalkenyl groups useful in the presentinvention include, but are not limited to, cyclopentenyl, cyclohexenyl,cycloheptenyl and cyclooctenyl. Bicycloalkenyl groups are also useful inthe present invention.

As used herein, the term “decreased expression” refers to the level of agene expression product that is lower and/or the activity of the geneexpression product is lowered. Preferably, the decrease is at least 20%,more preferably, the decrease is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, or at least 90% and mostpreferably, the decrease is at least 100%, relative to a control. Apreferred gene expression product for decreasing its expression isinterleukin-1 beta (IL-1β).

As used herein, the terms “derivative” or “derivatized” or “modified”refer to a compound that is produced from another compound of similarstructure by the replacement of substitution of one atom, molecule orgroup by another. For example, a hydrogen atom of a compound may besubstituted by alkyl, acyl, amino, hydroxyl, halo, haloalkyl, etc. toproduce a derivative of that compound or a derivatized compound. Thederivative of a compound preferably retains at least one function of thecompound from which it is produced, such as diminishing, abolishing orinterfering with the physiological action of a caspase-1 polypeptide.The activity of derivative compounds is tested as described herein.

By “determining the functional effect” is meant assaying for a compoundthat decreases a parameter that is indirectly or directly under theinfluence of a caspase-1 polypeptide, e.g., functional, enzymatic,physical and chemical effects. Such functional effects can be measuredby any means known to those skilled in the art, e.g., changes inspectroscopic characteristics (e.g., fluorescence, absorbance,refractive index), hydrodynamic (e.g., shape), chromatographic, orsolubility properties for the polypeptide, measuring inducible markersor activation and/or activity of the caspase-1 polypeptide; measuringbinding activity, e.g., binding of a compound to the caspase-1polypeptide, measuring cellular proliferation, measuring apoptosis,pyroptosis, or the like. The functional effects can be evaluated by manymeans known to those skilled in the art, e.g., microscopy forquantitative or qualitative measures of alterations in morphologicalfeatures, measurement of changes in caspase-1 RNA or protein levels,measurement of RNA stability, identification of downstream or reportergene expression (CAT, luciferase, β-gal, GFP and the like), e.g., viachemiluminescence, fluorescence, colorimetric reactions, antibodybinding, inducible markers, and ligand binding assays. “Functionaleffects” include in vitro, in vivo, and ex vivo activities.

As used herein, the term “different” means not the same, not of the sameidentity.

As used herein, “disorder”, “disease” or “pathological condition” areused inclusively and refer to any deviation from the normal structure orfunction of any part, organ or system of the body (or any combinationthereof). A specific disease is manifested by characteristic symptomsand signs, including biological, chemical and physical changes, and isoften associated with a variety of other factors including, but notlimited to, demographic, environmental, employment, genetic andmedically historical factors. Certain characteristic signs, symptoms,and related factors can be quantitated through a variety of methods toyield important diagnostic information. Disease specifically includesHIV-1 infection, AIDS and pathological conditions associated with ordeveloping in a subject as a consequence of HIV-1 infection and AIDS.

As used herein, “HAART” refers to a treatment for HIV-1 infection, whichis a cocktail of anti-viral drugs known as Highly Active Anti-RetroviralTherapy. HAART includes two reverse transcriptase inhibitors and aprotease inhibitor. HAART reduces the viral load in many patients tolevels below the current limits of detection, but the rapid mutationrate of this virus limits the efficacy of this therapy (Perrin andTelenti, 1998, Science 280:1871-1873).

As used herein, the term “haloalkyl” refers to alkyl groups substitutedby one or more halogen atoms, which may be the same or different.Exemplary, non-limiting, haloalkyl groups include CF₃, CCl₃, CHF₂,CHCl₂, C₂F₅,C₂Cl₅, and the like.

As used herein, the terms “halo” or “halogen,” by themselves or as partof another substituent, mean, unless otherwise stated, a fluorine (F),chlorine (Cl), bromine (Br), or iodine (I) atom.

As used herein, the term “heteroalkyl,” by itself or in combination withanother term, means, unless otherwise stated, a stable straight orbranched chain, or cyclic hydrocarbon radical, or combinations thereof,consisting of at least one carbon atoms and at least one heteroatomselected from the group consisting of O, N, P, Si and S, and wherein thenitrogen and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to twoheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂ CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′represents both —C(O)₂R′ and —R′C(O)₂. As described above, heteroalkylgroups, as used herein, include those groups that are attached to theremainder of the molecule through a heteroatom, such as —C(O)R′,—C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” isrecited, followed by recitations of specific heteroalkyl groups, such as—NR′R″ or the like, it will be understood that the terms heteroalkyl and—NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

As used herein, the term “hetero-biaryl” (when used as a group or aspart of a group) refers to a biaryl group containing the specifiednumber of atoms and which contains one or more nitrogen, sulphur, oroxygen heteroatoms. Examples of hetero-biaryl as used herein include,but are not limited to; quinoline, isoquinoline, quinoxaline, andbenzotriazine groups. Said hetero-biaryl groups may be optionallysubstituted. In some embodiments, the substitutions may be one or moregroups selected from C₁-C₃alkyl, C₁-C₃alkoxy, —C(O)Me, CO₂H, CO₂Me and═O.

As used herein, the term “heterocyclyl” (Hetcy) unless otherwisespecified, means mono- and bicyclic saturated rings and ring systemscontaining at least one heteroatom selected from N, S and O, each ofsaid ring having from 3 to 10 atoms in which the point of attachment maybe carbon or nitrogen. Examples of “heterocyclyl” include, but are notlimited to, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl,imidazolidinyl, tetrahydrofuranyl, 1,4-dioxanyl, morpholinyl,thiomorpholinyl, tetrahydrothienyl and the like. Heterocyclys can alsoexist in tautomeric forms, e.g., 2- and 4-pyridones. Heterocyclylmoreover includes such moieties in charged form, e.g., piperidinium.

In each of the above embodiments designating a number of atoms e.g.“C₁₋₈” is meant to include all possible embodiments that have one feweratom. Non-limiting examples include C₁₋₇, C₂₋₈, C₂₋₇, C₃₋₈, C₃₋₇ and thelike.

As used herein, “HIV” is used herein to refer to the humanimmunodeficiency virus. It is recognized that the HIV virus is anexample of a hyper-mutable retrovirus, having diverged into two majorsubtypes (HIV-1 and HIV-2), each of which has many subtypes.

As used herein, the term “HIV-1 infection” refers to indications of thepresence of the Human Immunodeficiency Virus type-1 (HIV-1) in anindividual and includes asymptomatic seropositivity, aids-relatedcomplex (arc), and acquired immunodeficiency syndrome (AIDS).

As used herein, the term “HIV-1 viral load” refers to the number ofHIV-1 viral particles in a sample of blood plasma. HIV-1 viral load isincreasingly employed as a surrogate marker for disease progression. Itis measured by PCR and bDNA tests and is expressed in number of HIV-1copies or equivalents per milliliter.

As used herein, the term “immune response” means any physiologicalchange resulting in activation and/or expansion of a “B” cell populationwith production of antibodies, and/or activation and/or expansion of a“T” cell population.

As used herein, the term “incomplete HIV-1 nucleic acid” refers to a notfull-length HIV-1 nucleic acid. Full-length HIV-1 nucleic acids haveabout 9,700-9,800 nt (single-stranded) or about 9,700-9,800 bps(double-stranded). Thus, an “incomplete HIV-1 nucleic acid” is an HIV-1nucleic acid of less than about 9,000 nt (single-stranded) or less thanabout 9,000 bps (double-stranded). Incomplete HIV-1 nucleic acids in acell can be the result of an abortive HIV-1 reverse transcriptionreaction.

As used herein, the terms “individual,” “subject,” “host,” and “patient”(used interchangeably herein), refer to a mammal, including, but notlimited to, humans and non-human mammals, such as simians. Preferred isa human. As used herein, “subject” or “patient” to be treated for apathological condition, disorder, or disease by a subject method meanseither a human or non-human mammal in need of treatment for apathological condition, disorder, or disease. The term “non-humanmammal” includes non-human primates (particularly higher primates),sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat,rabbits, cow, etc. In some embodiments, the subject is a human. In otherembodiments, the subject is an experimental animal or an animal suitableas a disease model.

As used herein, the terms “inhibition” or “inhibits” mean to reduce anactivity as compared to a control (e. g. an activity in the absence ofsuch inhibition). It is understood that inhibition can mean a slightreduction in activity to the complete ablation of all activity. An“inhibitor” can be anything that reduces activity. For example, aninhibition of caspase-1 activity by a disclosed composition can bedetermined by assaying the amount of IL-1β in the presence of thecomposition to the amount of IL-1β in the absence of the composition. Inthis example, if the amount of IL-1β is reduced in the presence of thecomposition as compared to the amount of IL-1β in the absence of thecomposition, the composition can be said to inhibit the activity ofcaspase-1. Inhibition of caspase-1 polypeptide activity is achieved whenthe level or activity value relative to a control is reduced by about10%, preferably about 20%, preferably about 30%, preferably about 40%,preferably about 50%, preferably about 60%, preferably about 70%,preferably about 80%, or preferably about 90-100%. Likewise, inhibitionof cell death is achieved when the cell death relative to a control isreduced by about 10%, preferably about 20%, preferably about 30%,preferably about 40%, preferably about 50%, preferably about 60%,preferably about 70%, preferably about 80%, or preferably about 90-100%.Likewise, inhibition of pyroptosis is achieved when pyroptosis relativeto a control is reduced by about 10%, preferably about 20%, preferablyabout 30%, preferably about 40%, preferably about 50%, preferably about60%, preferably about 70%, preferably about 80%, or preferably about90-100%.

As used herein, the term “in vitro” means outside the body of theorganism from which a cell or cells is obtained or from which a cellline is isolated.

As used herein, the term “in vivo” means within the body of the organismfrom which a cell or cells is obtained or from which a cell line isisolated.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” “Stereoisomer” and“stereoisomers” refer to compounds that exist in differentstereoisomeric forms if they possess one or more asymmetric centers or adouble bond with asymmetric substitution and, therefore, can be producedas individual stereoisomers or as mixtures. Stereoisomers includeenantiomers and diastereomers. Stereoisomers that are not mirror imagesof one another are termed “diastereomers” and those that arenon-superimposable mirror images of each other are termed “enantiomers.”When a compound has an asymmetric center, for example, it is bonded tofour different groups, a pair of enantiomers is possible. An enantiomercan be characterized by the absolute configuration of its asymmetriccenter and is described by the R- and S-sequencing rules of Cahn andPrelog, or by the manner in which the molecule rotates the plane ofpolarized light and designated as dextrorotatory or levorotatory (i.e.,as (+) or (−)-isomers respectively). A chiral compound can exist aseither individual enantiomer or as a mixture thereof. A mixturecontaining equal proportions of the enantiomers is called a “racemicmixture”. Unless otherwise indicated, the scope of the present inventionincludes individual enantiomers, racemates, diastereomers, tautomers,geometric isomers, and stereoisomers as well as mixtures of thecompounds. The methods for the determination of stereochemistry and theseparation of stereoisomers are well-known in the art (see discussion inChapter 4 of ADVANCED ORGANIC CHEMISTRY, 4th edition J. March, JohnWiley and Sons, New York, 1992) differ in the chirality of one or morestereocenters.

The terms “optional” or “optionally” as used throughout thespecification means that the subsequently described event orcircumstance may but need not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. For example, “heterocyclo group optionally mono- ordi-substituted with an alkyl group” means that the alkyl may, but neednot, be present, and the description includes situations where theheterocyclo group is mono- or di-substituted with an alkyl group andsituations where the heterocyclo group is not substituted with the alkylgroup. The terms also refer to a subsequently described composition thatmay but need not be present, and that the description includes instanceswhere the composition is present and instances in which the compositionis nor present.

As used herein, the term “pharmaceutically acceptable” refers tocompositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction when administered to asubject, preferably a human subject. Preferably, as used herein, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof a Federal or state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

As used herein, the term “physiologically functional derivative” refersto any pharmaceutically acceptable derivative of a compound of thepresent invention, for example an ester or an amide thereof, andincludes any pharmaceutically acceptable salt, ester, or salt of suchester of a compound of the present invention which, upon administrationto a mammal, such as a human, is capable of providing (directly orindirectly) a compound of the present invention or an active metaboliteor residue thereof. It will be appreciated by those skilled in the artthat the compounds of the present invention may be modified to providephysiologically functional derivatives thereof at any of the functionalgroups in the compounds, and that the compounds of the present inventionmay be so modified at more than one position.

As used herein, the terms “polypeptide” and “protein” (usedinterchangeably herein) refer to a polymer of amino acid residues.Preferred polypeptides are caspase-1 polypeptides, in particular humancaspase-1 polypeptides.

As used herein, the term “population of cells” refers to cells,preferably mammalian cells, more preferably human cells, grown in vitroor in vivo. The term also refers to cells within a host and may comprisea mixture of cells, such as virally infected cells and uninfected cells.A preferred population of cells is a population of CD4 T-cells. A morepreferred population of cells is a population of CD4 T-cells within ahost. An even more preferred population of cells is a population of CD4T-cells within a host comprising HIV-1 infected CD4 T-cells anduninfected CD4 T-cells.

As used herein, the term “preventing death of a cell in a population ofcells” or grammatical equivalents thereof means that in a population ofcells more cells survive when contacted with a compound of the inventionas compared to a cells in a population of cells not contacted with thecompound, but otherwise treated comparably (control). As such, the deathof a cell is prevented, when at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90-100% of cells in a population of cells survivewhen contacted with a compound of the invention as compared to cells ina population of cells not contacted with the compound, but otherwisetreated comparably (control).

As used herein, the term “prodrug” refers to a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic tocells compared to the parent drug and is capable of being enzymaticallyactivated or converted into the more active parent form. See, e.g.,Wihnan, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stellaet al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al, (ed.), pp. 247-267, HumanaPress (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,(3-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5 fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug.

As used herein, the term “protease inhibitor” (“PI”) refers toinhibitors of the HIV-1 protease, an enzyme required for the proteolyticcleavage of viral polyprotein precursors (e.g., viral GAG and GAG Polpolyproteins), into the individual functional proteins found ininfectious HIV-1. HIV protease inhibitors include compounds having apeptidomimetic structure, high molecular weight (7600 Daltons) andsubstantial peptide character, e.g. CRIXIVAN® (available from Merck) aswell as non-peptide protease inhibitors e.g., VIRACEPT® (available fromAgouron.

As used herein, the terms “salt” and “pharmaceutically acceptable salt”refer to salts of a compound which is prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,citric, tartaric, methanesulfonic, and the like. Also included are saltsof amino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge et al., 1977, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 66:1-19). Certain specific compounds of the present inventioncontain both basic and acidic functionalities that allow the compoundsto be converted into either base or acid addition salts. The neutralforms of a compound may be regenerated by contacting the salt with abase or acid and isolating the parent compound in the conventionalmanner. The parent form of the compound differs from the various saltforms in certain physical properties, such as solubility in polarsolvents, but otherwise the salts are equivalent to the parent form ofthe compound for the purposes of the present invention.

As used herein, the term “solvate” refers to a compound that iscomplexed to a solvent. Solvents that can form solvates with thecompounds of the present invention include common organic solvents suchas alcohols (methanol, ethanol, etc.), ethers, acetone, ethyl acetate,halogenated solvents (methylene chloride, chloroform, etc.), hexane andpentane. Additional solvents include water. When water is the complexingsolvent, the complex is termed a “hydrate.”

Each of the chemical terms herein (e.g., “alkyl,” “heteroalkyl,” “aryl”and “heteroaryl”) is meant to include both “unsubstituted” andoptionally “substituted” forms of the indicated radical, unlessotherwise indicated. Typically each radical is substituted with 0, 1, 23 4 or 5 substituents, unless otherwise indicated. Examples ofsubstituents for each type of radical are provided herein.

“Substituted” refers to a group as defined herein in which one or morebonds to a carbon(s) or hydrogen(s) are replaced by a bond tonon-hydrogen and non-carbon atom “substituents” such as, but not limitedto, a halogen atom such as F, Cl, Br, and I; an oxygen atom in groupssuch as hydroxyl groups, alkoxy groups, aryloxy, and acyloxy groups; asulfur atom in groups such as thiol groups, alkyl and aryl sulfidegroups, sulfone groups, sulfonyl groups, and sulfoxide groups; anitrogen atom in groups such as amino, alkylamines, dialkylamines,arylamines, alkylarylamines, diarylamines, alkoxyamino, hydroxyamino,acylamino, sulfonylamino, N-oxides, imides, and enamines; and otherheteroatoms in various other groups. “Substituents” also include groupsin which one or more bonds to a carbon(s) or hydrogen(s) atom isreplaced by a higher-order bond (e.g., a double or triple-bond) to aheteroatom such as oxygen in oxo, acyl, amido, alkoxycarbonyl,aminocarbonyl, carboxyl, and ester groups; nitrogen in groups such asimines, oximes, hydrazones, and nitriles. “Substituents” further includegroups in which one or more bonds to a carbon(s) or hydrogen(s) atoms isreplaced by a bond to a cycloalkyl, heterocyclyl, aryl, and heteroarylgroups. Representative “substituents” include, among others, groups inwhich one or more bonds to a carbon or hydrogen atom is/are replaced byone or more bonds to fluoro, chloro, or bromo group. Anotherrepresentative “substituent” is the trifluoromethyl group and othergroups that contain the trifluoromethyl group. Other representative“substituents” include those in which one or more bonds to a carbon orhydrogen atom is replaced by a bond to an oxygen atom such that thesubstituted alkyl group contains a hydroxyl, alkoxy, or aryloxy group.Other representative “substituents” include alkyl groups that have anamine, or a substituted or unsubstituted alkylamine, dialkylamine,arylamine, (alkyl)(aryl)amine, diarylamine, heterocyclylamine,diheterocyclylamine, (alkyl)(heterocyclyl)amine, or(aryl)(heterocyclyl)amine group. Still other representative“substituents” include those in which one or more bonds to a carbon(s)or hydrogen(s) atoms is replaced by a bond to an alkyl, cycloalkyl,aryl, heteroaryl, or heterocyclyl group.

As used herein, the terms “treatment”, “treating” or grammaticalequivalents thereof refer to both therapeutic treatment and prophylacticor preventative measures. Those in need of treatment include thosealready with the disease or disorder as well as those in which thedisease or disorder is to be prevented. Hence, a subject may have beendiagnosed as having the disease or disorder or may be predisposed orsusceptible to the disease. As such, the terms include: (1) preventing apathological condition, disorder, or disease, i.e. causing the clinicalsymptoms of a pathological condition, disorder, or disease not todevelop in a subject that may be predisposed to the pathologicalcondition, disorder, or disease but does not yet experience any symptomsof the pathological condition, disorder, or disease; (2) inhibiting thepathological condition, disorder, or disease, i.e. arresting or reducingthe development of the pathological condition, disorder, or disease orits clinical symptoms; or (3) relieving the pathological condition,disorder, or disease, i.e. causing regression of the pathologicalcondition, disorder, or disease or its clinical symptoms. These termsencompass also prophylaxis, therapy and cure. Treatment means any mannerin which the symptoms of a pathological condition, disorder, or diseaseare ameliorated or otherwise beneficially altered. Preferably, thesubject in need of such treatment is a mammal, more preferable a human.

II. Treatment of HIV-1 Infection and AIDS

Applicants have studied the mechanisms by which HIV kills helper CD4T-cells—the fundamental problem in AIDS. Using a physiologicallyrelevant system formed of human lymphoid tissues, it was demonstratedthat killing of CD4 T-cells by HIV can be efficiently prevented byinhibitors of caspase-1. Applicants' finding forms a new strategy forpreserving CD4 T-cells in AIDS patients.

Despite the vigorous research over the last 30 years, the cause of CD4T-cell death in AIDS remains poorly understood, and is cited as one ofthe top unsolved problems in HIV research. Applicants explored themechanisms by which HIV depletes CD4 T-cells using a unique,physiologically relevant experimental system formed of fresh humanlymphoid cultures. In many regards, this system is one of the mostpowerful experimental approaches to modeling molecular and cellularevents during HIV infection in human patients. Surprisingly, using thissystem Applicants discovered that the overwhelming majority of the cellsdie not from HIV, but rather from the cell's own defensive response toHIV before the virus can make copies of itself. After HIV-1infection, >95% of lymphoid CD4 T-cells that die are not productivelyinfected, and accumulate cytoplasmic viral DNA due to incomplete reversetranscription It appears that HIV enters the CD4 T-cells that aredestined to die and starts to make a DNA copy of its RNA, a processcalled reverse transcription. However, during this process, the cellssense the incomplete DNA intermediates (incomplete HIV nucleic acids)that accumulate in the cells are sensed and trigger the cell to ‘commitsuicide’ in an attempt to protect the body (Doitsh et al., 2010, Cell,143(5):789-801; incorporated herewith by reference). While this responseis likely designed to be protective, HIV subverts and amplifies it soeffectively that it becomes a central driver of HIV pathogenesis.

A second surprise was finding that the mechanism of cell death was not asilent one. These infected cells die a fiery death known as pyroptosis,causing significant inflammation as they erupt their cellular contentsand release chemical signals, which recruit healthy CD4 T-cells to thesite of infection. This establishes a vicious cycle, whereby the dyingCD4 T-cells release inflammatory signals that attract more cells to die(Doitsh et al., 2010, Cell, 143(5):789-801; incorporated herewith byreference).

Destruction of cells by pyroptosis also releases high levels of anintracellular component called adenosine-5′-triphosphate (5′ATP) intothe extracellular space. Extracellular ATP binds to membrane channelstermed P2X7 purinergic receptors and acts as an inflammatory stimulus toinduce pyroptosis in nearby cells. Thus, pyroptosis activated initiallyby HIV infection may result in an avalanche of new rounds of pyroptosisin healthy CD4 T-cells by the repeated release of intracellular ATP in avirus-independent manner (FIG. 1).

Lymphoid tissues serve as home to more than 98% of the body's CD4T-cells and form the primary sites of HIV replication and spread.Applicants' experimental system, built on these tissues, closelyrecapitulates these conditions and thus provides a compellingexperimental platform for studying HIV pathogenesis and exploring newstrategies aimed at blocking CD4 T-cell death thereby curbing AIDSprogression (Doitsh et al., 2010, Cell, 143(5):789-801; incorporatedherewith by reference).

The data provided herein suggest that CD4 T-cell depletion in AIDS isnot triggered by HIV-1 toxicity, but is mediated by a cellular antiviralinnate immune response against the virus. This response involves intenseinflammation associated with the pyroptotic death of CD4 T-cells, andwas likely designed to protect the host. However, the ensuinginflammation that results during this process may spin out of controlwith inflammation attracting new CD4 T-cells to undergo new rounds ofinfection and cell death.

The finding that CD4 T-cell death is associated with inflammationprovides an unexpected and exciting nexus between the virus and hostwith strong implications for the role of inflammation in HIVpathogenesis and disease progression. It is striking that SimianImmunodeficiency Virus (SIV) infection of its natural monkey hosts doesnot result in AIDS. While the SIV virus is cytopathic for CD4 T-cellslike HIV-1 (i.e., it also kills monkeys' CD4 T-cells), monkeys do notmount an inflammatory response when SIV infection occurs, as it does inhumans. Thus, from an evolutionary point of view, the threat of AIDS hasbeen neutralized not by controlling the virus but by negating aninflammatory response by the host against the virus. The discoverydescribed herein, namely that peptide and non-peptide caspase-1antagonists can be used to block the inflammation occurring during CD4T-cell pyroptosis and thus interrupt the generation of inflammatorysignals is surprising and unexpected. Thus, the peptide and non-peptidecaspase-1 antagonists described herein or related drugs open the door touse of an entirely new class of “anti-AIDS” agents that curb CD4 T-cellloss by suppressing pyroptosis and the associated inflammatory responsethat is pivotal in HIV-1 pathogenesis.

III. Compounds

Applicants discovered that small molecule compounds (peptide andnon-peptide small molecules compounds), that inhibit activation and/oractivity of caspase-1 are useful in the methods of the presentinvention, in particular, in methods for the treatment of a patienthaving an HIV-1 infection or suspected of having an HIV-1 infection orhaving AIDS and in methods for preventing the death of a CD4 T-cell in apopulation of CD4 T-cells comprising HIV-1 infected and uninfected CD4T-cells.

A. Caspase-1 Inhibitors

The present invention describes a variety of caspase-1 inhibitors foruse in the methods of the present invention. More specifically, thepresent invention describes a variety of caspase-1 inhibitors for use ascompositions and practicing methods of the present invention.

Caspase-1 inhibitors are currently commercially available from a numberof sources, including Calbiochem (La Jolla, Calif.), Biomol (Plymouth,Mass.), Sigma-Aldrich (St. Louis, Mo.), and A.G. Scientific, Inc.(caspases.com division) (San Diego, Calif.). In addition, a number ofexperimental caspase inhibitors are under development at a number ofpharmaceutical companies. The most commonly employed caspase inhibitorsare short-chain peptides (typically 3-6 amino acids), either per se ormodified with, for example, ester or carbonyl-containing groups. Typicalderivatives include aldehydes, chloromethyl ketones, fluoromethylketones, trifluoroacetates, benzoyloxymethyl ketones, nitroanilides, andvarious substituted coumarins. Lists of these are found, for example, inthe catalogs of the commercial sources mentioned above. These caspaseinhibitors are useful for practicing the present invention.

In some embodiments of the present invention, a caspase-1 inhibitor is apeptide caspase-1 inhibitor or single stereoisomers, mixtures ofstereoisomers, pharmaceutically acceptable salts or prodrugs thereof.

It is possible to generate reversible or irreversible inhibitors ofcaspase-1 activation by coupling caspase-1-specific peptides to certainaldehyde, nitrile or ketone compounds. These caspase inhibitors cansuccessfully inhibit the induction of apoptosis in various tumor celllines (Schlegel et al., J Biol Chem (1996) 271:1841; Martins et al., JBiol Chem (1997) 272:7421; Huang et al., Mol Cell Biol (1999) 19:2986;Guo and Kyprianou, Cancer Res (1999) 59:1366) as well as normal cells(Zaks et al., J Immunol (1999) 162:3273; Gastman et al., Cancer Res(1999) 59:1422). Peptide caspase-1 inhibitors described herein can bederivatized and act as effective irreversible inhibitors with noapparent added cytotoxic effect. Peptide caspase-1 inhibitors can bederivatized to include fluoromethyl ketone (fmk), tetra fluoro phenoxymethyl ketone (tfpmk) or an aldehyde group at the C-terminus. Peptidecaspase-1 inhibitors can also be synthesized with a benzyloxycarbonylgroup (known as BOC or Z) or an acetyl group at the N-terminus and withO-methyl side chains. They exhibit enhanced cellular permeability, thusgreatly facilitating their use in both in vitro cell culture as well asin vivo animal and human studies.

In some embodiments of the present invention, a peptide caspase-1inhibitor is selected from the group consisting of BACMK(Boc-Asp(Obzl)-CMK, z-VAD (Z-Val-Ala-Asp), BocD, LY333531, casputin,Ac-DQMD-CHO (Ac-Asp-Met-Gln-Asp-CHO) (SEQ ID NO: 3), CV-1013, VX-740,VX-765, VX-799, Ac-YVAD-CMK (SEQ ID NO: 4), IDN-5370, IDN-6556,IDN-6734, IDN-1965, IDN-1529, z-VAD-fmk (Z-Val-Ala-Asp(OMe)-Fluoromethyl ester), z-DEVD-cmk (SEQ ID NO: 5), Z-DEVD (SEQ ID NO: 6),Ac-YVAD-fmk (SEQ ID NO: 7), z-Asp-Ch2-DCB, Ac-IETD (SEQ ID NO: 8),Ac-VDVAD (SEQ ID NO: 9), Ac-DQMD (SEQ ID NO: 10), Ac-LEHD (SEQ ID NO:11), Z-WEHD (SEQ ID NO: 12), Z-WEHD-fmk (SEQ ID NO: 13),Z-WE(OMe)HD(OMe)-fmk (SEQ ID NO: 14), Z-YVAD (SEQ ID NO: 15), Z-YVAD-fmk(SEQ ID NO: 16), Ac-YVAD-cmk (SEQ ID NO: 17), Ac-VEID (SEQ ID NO: 18)and single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof.

In some embodiments of the present invention, a peptide caspase-1inhibitor is selected from the group consisting of Boc-Phg-Asp-fmk,Boc-(2-F-Phg)-Asp-fmk, Boc-(F₃-Val)-Asp-fmk, Boc-(3-F-Val)-Asp-fmk,Ac-Phg-Asp-fmk, Ac-(2-F-Phg)-Asp-fmk, Ac-(F₃-Val)-Asp-fmk,Ac-(3-F-Val)Asp-fmk, Z-Phg-Asp-fmk Z-(2-F-Phg)-Asp-fmk,Z-(F₃-Val)-Asp-fmk, Z-Chg-Asp-fmk, Z-(2-Fug)-Asp-fmk,Z-(4-F-Phg)-Asp-fmk, Z-(4-Cl-Phg)-Asp-fmk, Z-3-Thg)-Asp-fmk,Z-(2-Fua)-Asp-fmk, Z-(2-Tha)-Asp-fmk, Z-3-Fua)-Asp-fmk,Z-(3-Tha)-Asp-fmk, Z-(3-Cl-Ala)-Asp-fmk, Z-(3-F-Ala)-Asp-fmk,Z-(F₃-Ala)-Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fmk, Z-(3-C₁₋₃-F-Ala)-Asp-fmk,Z-(2-Me-Val)Asp-ink, Z-(2-Me-Ala)-Asp-fmk, Z-(2-i-Pr-β-Ala)-Asp-fmk,Z-(3-Ph-β-Ala)-Asp-fmk, Z-(3-CN-Ala)-Asp-fmk, Z-(1-Nal)-Asp-fmk,Z-Cha-Asp-fmk, Z-3-CF₃-Ala)Asp-fmk, Z-(4-CF₃-Phg)-Asp-fmk,Z-(3-Me₂N-Ala)-Asp-fmk, Z-(2-Abu)-Asp-ink, Z-Tle-Asp-fmk, Z-Cpg-Asp-fmk,Z-Cbg-Asp-fmk, Z-Thz-Asp-fmk, Z-(3-F-Val)-Asp-fmk, Z-2-Thg)Asp-fmk, andsingle stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof.

In some embodiments of the present invention, a peptide caspase-1inhibitor is Z-VAD or Z-VAD-fmk or single stereoisomers, mixtures ofstereoisomers, pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments of the present invention, a peptide caspase-1inhibitor is Z-WEHD (SEQ ID NO: 12), Z-WEHD fmk (SEQ ID NO: 13), orZ-WE(OMe)HD(OMe)-fmk (SEQ ID NO: 14), or single stereoisomers, mixturesof stereoisomers, pharmaceutically acceptable salts or prodrugs thereof.This caspase-1 inhibitor is available, e.g., from R&D Systems (catalognumber FMK002).

In some embodiments of the present invention, a peptide caspase-1inhibitor is Z-YVAD (SEQ ID NO: 15), Z-YVAD-fmk (SEQ ID NO: 16) orZ-YVAD(OMe)-fmk (SEQ ID NO: 20) or single stereoisomers, mixtures ofstereoisomers, pharmaceutically acceptable salts or prodrugs thereof.This caspase-1 inhibitor is available, e.g., by Enzyme Systems(Livermore, Calif., USA) and R&D Systems (catalog number FMK005).

In some embodiments of the present invention, a peptide caspase-1inhibitor is Z-DEVD (SEQ ID NO: 6) or Z-DEVD-fmk (SEQ ID NO: 18) orsingle stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof. This caspase-1 inhibitor isavailable, e.g., by Enzyme Systems, Livermore, Calif., USA).

In some embodiments of the present invention, a caspase-1 inhibitor isAc-YVAD-CMK (SEQ ID NO: 17) or single stereoisomers, mixtures ofstereoisomers, pharmaceutically acceptable salts or prodrugs thereof.This caspase-1 inhibitor can be obtained, e.g., from Calbiochem (Prod.No. 400012).

Other caspase-1 inhibitors that can be used in the practice of theinvention include without limitation those described in WO93/05071,WO93/09135, WO93/14777, WO95/26958, WO95/29672, WO95/33751, WO95/35308,WO96/03982, WO96/30395, WO97/07805, WO97/08174, WO97/22618, WO97/22619,WO97/27220, WO98/11109, WO98/11129, WO98/16502, WO98/16504, WO98/16505,WO98/24804, WO98/24805, WO99/47545, WO01/90063, EP 519748 (U.Sequivalents are U.S. Pat. Nos. 5,430,128 and 5,434,248), EP 547699, EP618223, EP 623592 (U.S. equivalents are U.S. Pat. Nos. 5,985,838, and6,576,614), EP 623606 (U.S. equivalents are U.S. Pat. Nos. 5,462,939 and5,585,486), EP 628550 (U.S. equivalents are U.S. Pat. Nos. 5,585,357 and5,677,283), EP 644198, U.S. Pat. No. 5,430,128, U.S. Pat. No. 5,434,248,U.S. Pat. No. 5,462,939, U.S. Pat. No. 5,552,400, U.S. Pat. No.5,565,430, U.S. Pat. No. 5,585,357, U.S. Pat. No. 5,585,486, U.S. Pat.No. 5,622,967, U.S. Pat. No. 5,639,745, U.S. Pat. No. 5,656,627, U.S.Pat. No. 5,670,494, U.S. Pat. No. 5,677,283, U.S. Pat. No. 5,716,929,U.S. Pat. No. 5,739,279, U.S. Pat. No. 5,756,465, U.S. Pat. No.5,756,466, U.S. Pat. No. 5,798,247, U.S. Pat. No. 5,798,442, U.S. Pat.No. 5,834,514, U.S. Pat. No. 5,843,904, U.S. Pat. No. 5,843,905, U.S.Pat. No. 5,847,135, U.S. Pat. No. 5,866,545, U.S. Pat. No. 5,843,904,U.S. Pat. No. 5,843,905, U.S. Pat. No. 5,847,135, U.S. Pat. No.5,866,545, U.S. Pat. No. 5,869,519, U.S. Pat. No. 5,874,424, U.S. Pat.No. 5,932,549, U.S. Pat. No. 7,417,029, US 2006/0128696, Mjalli et al.,1993, Bioorg Med Chem Lett 3:2689-2693, Mjalli et al., 1994, Bioorg MedChem Lett 4:1965-1968, Mjalli et al., 1995 Bioorg Med Chem Lett5:1405-1408, Mjalli et al., 1995, Bioorg Med Chem Lett 5:1409-1414,Thornberry et al., 1994, Biochem 33:3934-3940, Dolle et al., 1994, J MedChem 37:563-564, Dolle et al., 1994, J Med Chem 37: 3863-3866, Dolle etal., 1995, J Med Chem 38: 220-222, Graybill et al., 1997 Bioorg Med ChemLett 7:41-46, Semple et al., 1998, Bioorg Med Chem Lett 8:959-964, andOkamoto et al., 1999, Chem Pharm Bull 47:11-21, herewith incorporated byreference in their entireties for all purposes.

1. Caspase-1 Inhibitor Having Formula 1a or 1b

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2011/0144074, US2009/0215736, US2008/0015172, andUS2003/0092703 (herewith incorporated by reference in their entireties).Preferred compounds described in published US2011/0144074,US2009/0215736, US2008/0015172, and US2003/0092703 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 1a or 1b:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein:

next to R³ represents a single or double bond; Z is oxygen or sulfur; R¹is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y; R is a C₁₋₁₂aliphatic, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl; Y is anelectronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is a group capable of fitting into the S2 sub-siteof a caspase; R⁴ is hydrogen or a C₁₋₆ aliphatic group that isoptionally interrupted by —O—, —S—, —SO₂—, —CO—, —NH—, or —N(C₁₋₄alkyl)-, or R³ and R⁴ taken together with their intervening atomsoptionally form a 3-7 membered ring having 0-2 heteroatoms selected fromnitrogen, oxygen or sulfur; Ring A is a nitrogen-containing mono-, bi-or tricyclic ring system having 0-5 additional ring heteroatoms selectedfrom nitrogen, oxygen or sulfur; Ring B is a nitrogen-containing 5-7membered ring having 0-2 additional ring heteroatoms selected fromnitrogen, oxygen or sulfur; R⁵ is R⁶, (CH₂)_(n)R⁶, COR^(E), CO₂R⁶,SO₂R⁶, CON(R⁶)₂, or SO₂N(R⁶)₂; n is one to three; and each R⁶ isindependently selected from hydrogen, an optionally substituted C₁₋₄aliphatic group, an optionally substituted C₆₋₁₀ aryl group, or a mono-or bicyclic heteroaryl group having 5-10 ring atoms.

2. Caspase-1 Inhibitor Having Formula 2

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2011/0137037 and US2007/0155718 (herewith incorporated byreference in their entireties). Preferred compounds described inpublished US2011/0137037 and US2007/0155718 for use in the methods ofthe present invention are referred to herein as caspase-1 inhibitorhaving Formula 2:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is H, R⁴, haloalkyl,CHN₂, CH₂Cl, CH₂F, —CH₂OPO(R⁴)₂, —CH₂OPO(OR⁴)₂, or —C₁₋₂alkyl-R³—R⁴; R²is a P₄—P₃—P₂, P₃—P₂, or P₂ moiety of a caspase-1 inhibitor; R³ is —O—,—NH—, —NR⁴—, —S—, or —O(C═O)—; R⁴ is C₁₋₁₂aliphatic, C₆₋₁₀aryl, 5-10membered heterocyclyl, 5-10 membered heteroaryl, C₃₋₁₀cycloaliphatic,—(C₁₋₆alkyl)-C₆₋₁₀aryl, —(C₁₋₆ alkyl)-(5-10 membered heteroaryl), —(C₁₋₆alkyl)-(5-10 membered heterocyclyl), or —(C₁₋₆alkyl)-C₃₋₁₀cycloaliphatic; wherein said R⁴ group is optionallysubstituted with 0-5 J and 0-2 J²; or two R⁴ groups, together with theatom to which they are attached, form a 3-8 membered monocyclic or 8-12membered bicyclic ring optionally substituted with 0-5 J and 0-2 J²; Jis halogen, —OR′, —NO₂, —CN, —CF₃, —OCF₃, —R′, 1,2-methylenedioxy,1,2-ethylenedioxy, —N(R′)₂, —SR′, —SOR′, SO₂R′, —SO₂N(R′)₂, —SO₃R′,C(O)R′, —C(O)C(O)R′, —C(O)C(O)OR′, —C(O)C(O)N(R′)₂, —C(O)CH₂C(O)R′,—C(S)R′, —C(S)OR′, —C(O)OR′, —OC(O)R′, —C(O)N(R′)₂, —OC(O)N(R′)₂,—C(S)N(R′)₂, —(CH₂)₀₋₂NHC(O)R′, —N(R′)N(R′)COR′, —N(R′)N(R′)C(O)OR′,—N(R′)N(R′)CON(R′)₂, —N(R′)SO₂R′, —N(R′)SO₂N(R′)₂, —N(R′)C(O)OR′,—N(R′)C(O)R′, —N(R′)C(S)R′, —N(R′)C(O)N(R′)₂, —N(R′)C(S)N(R′)₂,—N(COR′)COR′, —N(OR′)R′, —CN, —C(═NR′)N(′R)₂, —C(O)N(OR′)R′,—C(═NOR′)R′, —OP(O)(OR′)₂, —P(O)(R′)₂, —P(O)(OR′)₂, or —P(O)(H)(OR′); J₂is ═NR′, ═N(OR′), ═O, or ═S; R′ is H, C₁₋₁₂aliphatic, C₆₋₁₀aryl, 5-10membered heterocyclyl, 5-10 membered heteroaryl, C₃₋₁₀ cycloaliphatic,—(C₁₋₆ alkyl)-C₆₋₁₀aryl, —(C₁₋₆ alkyl)-(5-10 membered heteroaryl),—(C₁₋₆ alkyl)-(5-10 membered heterocyclyl), or —(C₁₋₆alkyl)-C₃₋₁₀cycloaliphatic; each R′ is independently and optionallysubstituted with 0-5 occurrences of H, C₁₋₆alkyl, CF₃, halogen, NO₂,OCF₃, CN, OH, O(C₁₋₆alkyl), NH₂, N(C₁₋₆alkyl), N(C₁₋₆alkyl)₂, C(═O)CH₃,or C₁₋₆alkyl optionally interrupted 1 time with a heteroatom selectedfrom O, N, and S; wherein each C₁₋₆alkyl is unsubstituted; unlessotherwise indicated, any group with suitable valence is optionallysubstituted with 0-5 J and 0-2 J².

3. Caspase-1 Inhibitor Having Formula 3

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2011/0130436 and US2009/0131456 (herewith incorporated byreference in their entireties). Preferred compounds described inpublished US2011/0130436 and US2009/0131456 for use in the methods ofthe present invention are referred to herein as caspase-1 inhibitorhaving Formula 3:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, or CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving groupor —OR, —SR, —OC═O(R), or —OPO(R⁸)(R⁹); R⁸ and R⁹ are independentlyselected from R or OR; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is hydrogen or a C₁₋₆ straight chained or branchedalkyl; and R⁴ is independently selected from hydrogen, halo, R, OR, SR,aryl, substituted aryl, OH, NO₂, CN, NH₂, NHR, N(R)₂, NHCOR, NHCONHR,NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR, CON(R)₂, S(O)₂R,SONH₂, S(O)R, SO₂NHR, or NHS(O)₂R.

4. Caspase-1 Inhibitor Having Formula 4, 4.1, 4.2, Or 4.3

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2011/0077190 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2011/0077190 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having 4:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein a is 0 or 1; b is 0 or 1provided that when b is 0, a is 0; A is 1) H, 2) C₁-C₆ alkyl, 3) aryl,4) heteroaryl, 5) heterocyclyl, 6) R³—C(O)—, 7) R³—C(O)—; 8) R³—C(O)O—,or 9) R³—S(O)₂—; P2, P3, P4 and, when present, P5 and PX, are any (D) or(L) amino acid residue; the line “-” when located between P2, P3, P4, P5or PX represents a peptide bond or a peptidomimetic bond; the wavy linerepresents either cis or trans orientation of R¹ and R²; R¹ is 1) aryl,2) heteroaryl, 3) heterocyclyl, 4) C₂-C₆ alkene-R²⁰, 5) SO₂R⁵, 6) SO₃R⁵,7) SOR⁵, 8) SONHR⁵, 9) SO₂NHR⁵, 10) CN, 11) CO₂R⁵, 12) COR⁵, 13) PO₃R⁵,14) PO(OR⁵)₂, or 15) PO(OR⁵), wherein the aryl, the heteroaryl, or theheterocyclyl are optionally substituted with one or more R³⁰; R² is 1)R¹; or 2) H, 3) halogen, 4) haloalkyl, 5) C₁-C₆ alkyl, 6) C₂-C₆ alkene,7) C₃-C₇ cycloalkyl, 8) OR⁹; 9) OCOR⁶, 10) OCO₂R⁶, 11) NR⁷R⁸, 12)NHSO₂R⁶, 13) NHCOR⁶, 14) aryl, 15) heteroaryl, or 16) heterocyclyl; R³is 1) C₁-C₆ alkyl, 2) aryl, 3) heteroaryl, or 4) heterocyclyl; R⁴ is 1)H, or 2) C₁-C₆ alkyl; R⁵ is 1) H, 2) C₁-C₆ alkyl, 3) C₂-C₆ alkene, 4)C₃-C₇ cycloalkyl, 5) aryl, 6) heteroaryl, 7) heterocyclyl, or 8) anyoptionally protected (D) or (L) amino acid residue, or non-natural aminoacid residue; R⁶ is 1) any (D) or (L) amino acid residue or non-naturalamino acid residue, 2) C₁-C₆ alkyl, 3) C₃-C₇ cycloalkyl, 4) aryl, 5)heteroaryl, or 6) heterocyclyl, in which the alkyl or the cycloalkyl areoptionally substituted with one or more R¹⁰ substituents; and in whichthe aryl, heteroaryl or heterocyclyl are optionally substituted with oneor more R²⁰ substituents; R⁷ and R⁸ are independently selected from: 1)H, 2) C₁-C₆ alkyl, 3) C₃-C₇ cycloalkyl, 4) haloalkyl, 5) aryl, 6)heteroaryl, or 7) heterocyclyl, wherein the alkyl and the cycloalkyl areoptionally substituted with one or more R¹⁰ substituents, and the aryl,the heteroaryl and the heterocyclyl are optionally substituted with oneor more R²⁰ substituents; R⁹ is 1) H, 2) C₁-C₆ alkyl, 3) C₃-C₇cycloalkyl, 4) aryl, 5) heteroaryl, or 6) heterocyclyl, in which thealkyl or the cycloalkyl are optionally substituted with one or more R¹⁰substituents; and in which the aryl, heteroaryl or heterocyclyl areoptionally substituted with one or more R²⁰ substituents; R¹⁰ isindependently selected from: 1) halogen, 2) C₁-C₆ alkyl, 3) C₃-C₇cycloalkyl, 4) haloalkyl, 5) aryl, 6) heteroaryl, 7) heterocyclyl, 8)OR⁹, 9) S(O)_(m)R⁹, 10) NR⁷R⁸, 11) COR⁹, 12) C(O)OR⁹, 13) OC(O)R⁹, 14)SC(O)R⁹, 15) CONR⁷R⁸, or 16) S(O)₂NR⁷R⁸; R²⁰ is independently selectedfrom: 1) halogen, 2) NO₂, 3) CN, 4) C₁-C₆ alkyl, 5) haloalkyl, 6) C₃-C₇cycloalkyl, 7) OR⁷, 8) NR⁷R⁸, 9) SR⁷, 10) aryl, 11) heteroaryl, 12)heterocyclyl, 13) SO₂R⁵, 14) SO₃R⁵, 15) SOR⁵, 16) SONHR⁵, 17)SO₂NHR⁵,18) PO₃R⁵, 19) PO(OR⁵)₂, 20) PO(OR⁵), 21) COR⁷, 22) CO₂R⁷, 23)S(O)_(m)R⁷, 24) CONR⁷R⁸, or 25) S(O)₂NR⁷R⁸, wherein the alkyl and thecycloalkyl are optionally substituted with one or more R⁶ substituents;and wherein the aryl, the heteroaryl, or the heterocyclyl are optionallysubstituted with one or more R³⁰; R³⁰ is 1) NO₂, 2) C₂-C₆ alkene-R²⁰, 3)SO₂R⁵, 4) SOR⁵, 5) SONHR⁵, 6) SO₂NHR⁵, 7) CN, 8) CO₂R⁵, 9) COR⁵, 10)PO₃R⁵, 11) PO(OR⁵)₂, or 12) PO(OR⁵);or the compound is labeled with a detectable label or an affinity tagthereof, of Formula 4.1 (referred to herein as caspase-1 inhibitorhaving Formula 4.1):

wherein a is 0 or 1; b is 0 or 1 provided that when b is 0, a is 0; Ais 1) H, 2) C₁-C₆ alkyl, 3) aryl, 4) heteroaryl, 5) heterocyclyl, 6)R³—OC(O)—; 7) R³—C(O)O—, or 8) R³—S(O)₂—; AA₂ is the (R) or (S) aminoacid side chain of Val, Leu, Pro, Met, Ala, Thr, His, Ser, Lys, or Ile;AA₃ is the (R) or (S) amino acid side chain of Trp, Tyr, Ala, Asp, Gln,Glu, Phe, Ser, Thr, Val, Tyr, Gly, Leu, His, or Ile; or AA₃ isphenylglycine, indanylglycine, or Ala-(2′-quinolyl); AA₄ is the (R) or(S) amino acid side chain of Asp, Ile, Leu, Glu, Ala, Val, Tyr, Trp,Phe, or Pro; AA₅, when present, is the (R) or (S) amino acid side chainof Val or Leu; AA_(X), when present, is the (R) or (S) amino acid sidechain of any D or L amino acid residue or the amino acid side chain ofthe non-natural amino acid residue; the wavy line represents either cisor trans orientation of R¹ and R²; R¹ is 1) aryl, 2) heteroaryl, 3)heterocyclyl, 4) C₂-C₆ alkene-R²⁰, 5) SO₂R⁵, 6) SO₃R⁵, 7) SOR⁵, 8)SONHR⁵, 9) SO₂NHR⁵, 10) CN, 11) CO₂R⁵, 12) COR⁵, 13) PO₃R⁵, 14)PO(OR⁵)₂, or 15) PO(OR⁵), wherein the aryl, the heteroaryl, or theheterocyclyl are optionally substituted with one or more R³⁰; R² is 1)R¹; or 2) H, 3) halogen, 4) haloalkyl, 5) C₁-C₆ alkyl, 6) C₂-C₆ alkene,7) C₃-C₇ cycloalkyl, 8) OR⁹; 9) OCOR⁶, 10) OCO₂R⁶, 11) NR⁷R⁸, 12)NHSO₂R⁶, 13) NHCOR⁶, 14) aryl, 15) heteroaryl, or 16) heterocyclyl; R³is 1) C₁-C₆ alkyl, 2) aryl, 3) heteroaryl, or 4) heterocyclyl; R⁴ is 1)H, or 2) C₁-C₆ alkyl; R⁵ is 1) H, 2) C₁-C₆ alkyl, 3) C₂-C₆ alkene, 4)C₃-C₇ cycloalkyl, 5) aryl, 6) heteroaryl, 7) heterocyclyl, or 8) anyoptionally protected (D) or (L) amino acid residue; R⁶ is 1) any (D) or(L) amino acid residue, 2) C₁-C₆ alkyl, 3) C₃-C₇ cycloalkyl, 4) aryl, 5)heteroaryl, or 6) heterocyclyl, in which the alkyl or the cycloalkyl areoptionally substituted with one or more R¹⁰ substituents; and in whichthe aryl, heteroaryl or heterocyclyl are optionally substituted with oneor more R²⁰ substituents; R⁷ and R⁸ are independently selected from: 1)H, 2) C₁-C₆ alkyl, 3) C₃-C₇ cycloalkyl, 4) haloalkyl, 5) aryl, 6)heteroaryl, or 7) heterocyclyl, wherein the alkyl and the cycloalkyl areoptionally substituted with one or more R¹⁰ substituents, and the aryl,the heteroaryl and the heterocyclyl are optionally substituted with oneor more R²⁰ substituents; R⁹ is 1) H, 2) C₁-C₆ alkyl, 3) C₃-C₇cycloalkyl, 4) aryl, 5) heteroaryl, or 6) heterocyclyl, in which thealkyl or the cycloalkyl are optionally substituted with one or more R¹⁰substituents; and in which the aryl, heteroaryl or heterocyclyl areoptionally substituted with one or more R²⁰ substituents; R¹⁰ isindependently selected from: 1) halogen, 2) C₁-C₆ alkyl, 3) C₃-C₇cycloalkyl, 4) haloalkyl, 5) aryl, 6) heteroaryl, 7) heterocyclyl, 8)OR⁹, 9) S(O)mR⁹, 10) NR⁷R⁸, 11) COR⁵, 12) C(O)OR⁹, 13) OC(O)R⁹, 14)SC(O)R⁹, 15) CONR⁷R⁸, or 16) S(O)₂NR⁷R⁸; R²⁰ is independently selectedfrom: 1) halogen, 2) NO₂, 3) CN, 4) C₁-C₆ alkyl, 5) haloalkyl, 6) C₃-C₇cycloalkyl, 7) OR⁷, 8) NR⁷R⁸, 9) SR⁷, 10) aryl, 11) heteroaryl, 12)heterocyclyl, 13) SO₂R⁵, 14) SO₃R⁵, 15) SOR⁵, 16) SONHR⁵, 17) SO₂NHR⁵,18) PO₃R⁵, 19) PO(OR⁵)₂, 20) PO(OR⁵), 21) COR⁷, 22) CO₂R⁷, 23) S(O)mR⁷,24) CONR⁷R⁸, or 25) S(O)₂NR⁷R⁸, wherein the alkyl and the cycloalkyl areoptionally substituted with one or more R⁶ substituents; and wherein thearyl, the heteroaryl, or the heterocyclyl are optionally substitutedwith one or more R³⁰; R³⁰ is 1)NO₂, 2) C₂-C₆ alkene-R²⁰, 3) SO₂R⁵, 4)SOR⁵, 5) SONHR⁵, 6) SO₂NHR⁵, 7) CN, 8) CO₂R⁵, 9) COR⁵, 10) PO₃R⁵, 11)PO(OR⁵)₂, or 12) PO(OR⁵); or the compound is labeled with a detectablelabel or an affinity tag thereof;a compound of Formula 4.2 (referred to herein as caspase-1 inhibitorhaving Formula 4.2):

wherein AA₂ is the amino acid side chain of Val, Leu, Pro, Met, Ala,Thr, His, Ser, Lys, or Ile; AA₃ is the amino acid side chain of Trp,Tyr, Ala, Asp, Gln, Phe, Ser, Thr, Val, Tyr, Gly, Leu; or AA₃ isphenylglycine, indanylglycine, or Ala-(2′-quinolyl); AA₄ is the aminoacid side chain of Asp or Trp; or wherein AA₂ is the amino acid sidechain of Thr, His, Val, Trp, Ile, or Ala AA₃ is the amino acid sidechain of Glu or AA₃ is Ala-(2′-quinolyl); AA₄ is the amino acid sidechain of Ile, Leu, Glu, Asp, Ala, Pro or Val; or wherein AA, is theamino acid side chain of Val, Ala, Thr, or His; AA₃ is the amino acidside chain of Glu, Gln, Asp, Ala, Gly, Thr, Val, Trp; or AA₃ isphenylglycine or indanylglycine; AA₄ is the amino acid side chain ofTyr, Trp, Phe, or Asp;or a compound of Formula 4.3 (referred to herein as caspase-1 inhibitorhaving Formula 4.3):

wherein AA₂ is the amino acid side chain of Ala, Ser, Lys or Val; AA₃ isthe amino acid side chain of Val, Glu, Thr, or Gln; AA₄ is the aminoacid side chain of Asp, or Leu; AA₅ is the amino acid side chain of Valor Leu.

5. Caspase-1 Inhibitor Having Formula 5

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2011/0003824 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2011/0003824 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 5:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: R¹ is hydrogen, CHN₂, R,or —CH₂Y; R is an aliphatic group, an aryl group, an aralkyl group, aheterocyclic group, or a heterocyclylalkyl group; Y is group halogen,arylsulfonyloxy, alkylsulfonyloxy, trifluoromethanesulfonyloxy, OR, SR,—OC═O(R), or —OPO(R³)(R⁴); R² is CO₂H, CH₂CO₂H, or an ester of CO₂H orCH₂CO₂H selected from the group consisting of C₁₋₁₂aliphatic esters,aryl esters, aralkyl esters, heterocyclyl esters, heteroclyclylalkylesters; C₁₋₁₂aliphatic amides, aryl amides, aralkyl amides, keterocyclylamides, and heterocyclylalkyl amides; or R² is an isostere of CO₂H orCH₂CO₂H selected from the group consisting of CONHSO₂(alkyl) andCH₂CONHSO₂(alkyl); X₂—X₁ is C(R³)₂—C(R³), C(R³)═C, C(═O)—C(R³); each R³is independently selected from hydrogen or C₁₋₆ aliphatic, Ring C is afused aryl ring; n is 0, 1 or 2; and each methylene carbon in Ring A isoptionally and independently substituted by ═O, or by one or morehalogen, C₁₋₄ alkyl, or C₁₋₄ alkoxy.

6. Caspase-1 Inhibitor Having Formula 6

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2010/0137359 and US2004/0192612 (herewith incorporated byreference in their entireties). Preferred compounds described inpublished US2010/0137359 and US2004/0192612 for use in the methods ofthe present invention are referred to herein as caspase-1 inhibitorhaving Formula 6:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: X is —OR¹ or —N(R⁵)₂, Yis halo, trifluorophenoxy, or tetrafluorophenoxy; R¹ is: C₁₋₆ straightchained or branched alkyl, or C₂₋₆ straight chained or branched alkenylor alkynyl, wherein the alkyl, alkenyl, or alkynyl is optionallysubstituted with optionally substituted phenyl, CF₃, Cl, F, OMe, OEt,OCF₃, CN, or NMe₂; C₃₋₆ cycloalkyl, wherein 1-2 carbon atoms in thecycloalkyl is optionally replaced with —O— or —NR⁵—; R² is C₁₋₆ straightchained or branched alkyl; R³ is hydrogen, halo, OCF₃, CN, or CF₃; R⁴ ishydrogen, halo, OCF₃, CN, or CF₃; and each R⁵ is independently H, C₁₋₆straight chained or branched alkyl, aryl, —O—C₁₋₆ straight chained orbranched alkyl, or —O-aryl.

7. Caspase-1 Inhibitor Having Formula 7

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2010/0105914 and US2005/0233979 (herewith incorporated byreference in their entireties). Preferred compounds described inpublished US2010/0105914 and US2005/0233979 for use in the methods ofthe present invention are referred to herein as caspase-1 inhibitorhaving Formula 7:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein Y is

R¹ is H, C₁₋₁₂ aliphatic, C₃₋₁₀ cyclo aliphatic, C₆₋₁₀ aryl, 5-10membered heterocyclyl, 5-10 membered hetero aryl, (C₃₋₁₀ cycloalkyl)-(C₁₋₁₂ aliphatic)-, cyclo alkenyl-(C₁₋₁₂ aliphatic)-, (C₆₋₁₀aryl)-(C₁₋₁₂ aliphatic)-, (5-10 membered heterocyclyl)-(C₁₋₁₂aliphatic)-, or (5-10 membered heteroaryl)-(C₁₋₁₂aliphatic)-, whereinany hydrogen atom is optionally and independently replaced by R⁸ and anyset of two hydrogen atoms bound to the same atom is optionally andindependently replaced by carbonyl; Ring A is

wherein, in each ring, any hydrogen atom is optionally and independentlyreplaced by R⁴ and any set of two hydrogen atoms bound to the same atomis optionally and independently replaced by carbonyl; when Ring A is

then R is R³—C(O)—, HC(O), R³SO₂—, R³OC(O), (R³)₂NC(O), (R³)(H)NC(O),R³C(O)C(O)—, R³—, (R³)₂NC(O)C(O), (R³)(H)NC(O)C(O), or R³OC(O)C(O)—; andR³ is C₁₋₁₂aliphatic, C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl,(C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic)-, (C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-,(5-10 membered heterocyclyl)-(C₁₋₁₂aliphatic)-, or (5-10 memberedheteroaryl)-(C₁₋₁₂aliphatic)-; or two R³ groups bound to the same atomform together with that atom a 3-10 membered aromatic or nonaromaticring; wherein any ring is optionally fused to an C₆₋₁₀aryl, 5-10membered heteroaryl, C₃₋₁₀cycloalkyl, or 5-10 membered heterocyclyl;wherein up to 3 aliphatic carbon atoms may be replaced by a groupselected from O, N, NR⁹, S, SO, and SO₂, wherein R³ is substituted withup to 6 substituents independently selected from R⁸; when Ring A is

then R is R³—C(O)—, as shown below:

and R³ is phenyl, thiophene, or pyridine, wherein each ring isoptionally substituted with up to 5 groups independently selected fromR^(8′), and wherein at least one position on the phenyl, thiophene, orpyridine adjacent to bond x is substituted by R¹², wherein R¹² has nomore than 5 straight-chained atoms; R⁴ is halogen, —OR⁹, —NO₂—CN —CF₃,—OCF₁, —R⁹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁹)₂, —SR⁹, —SOR⁹,—SO₂R⁹—SO₂N(R⁹)₂, —SO₃R⁹, —C(O)R⁹, —C(O)C(O)R⁹, —C(O)C(O)OR⁹,—C(O)C(O)N(R⁹)₂, —C(O)CH₂C(O)R⁹, —C(S)R⁹, —C(S)OR⁹, —C(O)OR⁹, —OC(O)R⁹,—C(O)N(R⁹)₂, —OC(O)N(R⁹)₂, —C(S)N(R⁹)₂, —(CH₂)₀₋₂NHC(O)R⁹,—N(R⁹)N(R⁹)COR⁹, —N(R⁹)N(R⁹)C(O)OR⁹, —N(R⁹)N(R⁹)CON(R⁹)₂, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂N(R⁹)₂, —N(R⁹)C(O)OR⁹, —N(R⁹)C(O)R⁹, —N(R⁹)C(S)R⁹,—N(R⁹)C(O)N(R⁹)₂, —N(R⁹)C(S)N(R⁹)₂—N(COR⁹)COR⁹, —N(OR⁹)R⁹,—C(═NH)N(R⁹)₂, —C(O)N(OR⁹)R⁹, —C(═NOR⁹)R⁹, —OP(O)(OR⁹)₂, —P(O)(R⁹)₂,—P(O)(OR⁹)₂, or —P(O)(H)(OR⁹); R² is —C(R⁵)(R⁶)(R⁷), C₆₋₁₀aryl, 5-10membered heteroaryl, or C₃₋₇ cycloalkyl; R⁵ is H or a C₁₋₆straight-chained or branched alkyl; R⁶ is H or a C₁₋₆ straight-chainedor branched alkyl; R⁷ is —CF₃, —C₃₋₇cycloalkyl, C₆₋₁₀aryl, 5-10 memberedheteroaryl, heterocycle, or a C₁₋₆ straight-chained or branched alkyl,wherein each carbon atom of the alkyl is optionally and independentlysubstituted with R¹⁰; or R⁵ and R⁷ taken together with the carbon atomto which they are attached form a 3-10 membered cycloaliphatic; R⁸ andR^(8′) are each independently halogen, —OR⁹, —NO₂, —CN, —CF₃, —OCF₃,—R⁹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁹)₂, —SR⁹, —SOR⁹,—SO₂R⁹, —SO₂N(R⁹)₂—SO₃R⁹, —C(O)R⁹, —C(O)C(O)R⁹, —C(O)C(O)OR⁹,—C(O)C(O)N(R⁹)₂, —C(O)CH₂C(O)R⁹, —C(S)R⁹, —C(S)OR⁹, —C(O)OR⁹, —OC(O)R⁹,—C(O)N(R⁹)₂, —OC(O)N(R⁹)₂, —C(S)N(R⁹)₂, —(CH₂)₀₋₂NHC(O)R⁹,—N(R⁹)N(R⁹)COR⁹, —N(R⁹)N(R⁹)C(O)OR⁹, —N(R⁹)N(R⁹)CON(R⁹)₂, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂N(R⁹)₂, —N(R⁹)C(O)OR⁹, —N(R⁹)C(O)R⁹, —N(R⁹)C(S)R⁹,—N(R⁹)C(O)N(R⁹)₂, —N(R⁹)C(S)N(R⁹)₂, —N(COR⁹)COR⁹, —N(OR⁹)R⁹,—C(═NH)N(R⁹)₂, —C(O)N(OR⁹)R⁹, —C(═NOR⁹)R⁹, —OP(O)(OR⁹)₂, —P(O)(R⁹)₂,—P(O)(OR⁹)₂, and —P(O)(H)(OR⁹); R⁹ is hydrogen, C₁₋₁₂aliphatic,C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 membered heterocyclyl, 5-10membered heteroaryl, (C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic)-,(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or heteroaryl-(C₁₋₁₂aliphatic)-;wherein any hydrogen atom is optionally and independently replaced byR¹³ and any set of two hydrogen atoms bound to the same atom isoptionally and independently replaced by carbonyl; R¹⁰ is halogen,—OR¹¹, —NO₂, —CN, —CF₃—OCF₃, —R¹¹, or —SR¹¹; wherein R¹¹ isC₁₋₄-aliphatic-; R¹¹ is C₁₋₄-aliphatic-; R¹² is halogen, —OR¹¹,—NO₂—CN—CF₃—OCF₃, —R¹¹, or —SR⁹; R¹³ is —OR¹¹, —NO₂, —CN, —CF₃, —OCF₃,—R¹¹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R¹¹)₂, —SR¹¹, —SOR¹¹,—SO₇R¹¹—SO₂N(R¹¹)₂—SO₃R¹¹, —C(O)R¹¹, —C(O)C(O)R¹¹, —C(O)C(O)OR¹¹,—C(O)C(O)N(R¹¹)₂, —C(O)CH₂C(O)R¹¹—C(S)R¹¹, —C(S)OR¹¹, —C(O)OR¹¹,—OC(O)R¹¹, —C(O)N(R¹¹)₂, —OC(O)N(R¹¹)₂, —C(S)N(R¹¹)₂,—(CH₂)₀₋₂NHC(O)R¹¹, —N(R¹¹)N(R¹¹)COR¹¹, —N(R¹¹)N(R¹¹)C(O)OR¹¹,—N(R¹¹)N(R¹¹)CON(R¹¹)₂, —N(R¹¹)SO₂R¹¹, —N(R¹¹)SO₂N(R¹¹)₂,—N(R¹¹)C(O)OR¹¹, —N(R¹¹)C(O)R¹¹, —N(R¹¹)C(S)R¹¹, —N(R¹¹)C(O)N(R¹¹)₂,—N(R¹¹)C(S)N(R¹¹)₂, —N(COR¹¹)COR¹¹, —N(OR¹¹)R¹¹, —C(═NH)N(R¹¹)₂,—C(O)N(OR¹¹)R¹¹, —C(═NOR¹¹)R¹¹, —OP(O)(OR¹¹)₂, —P(O)(R¹¹)₂,—P(O)(OR¹¹)₂, and —P(O)(H)(OR¹¹); R¹¹ is hydrogen, C₁₋₁₂aliphatic, C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 membered heterocyclyl, 5-10 memberedheteroaryl, (C₃₋₁₂cycloaliphatic)-(C₁₋₁₂aliphatic),(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 membered heterocyclyl)-(C₁₋₁₂aliphatic)-, or heteroaryl-(C₁₋₁₂aliphatic)-; comprising reacting acompound shown below:

wherein R¹, R², and Ring A are as defined above; and a compound ofFormula RX, wherein R is as defined above and —X is OH or an appropriatederivative or leaving group, in the presence of conditions for couplingan amine and an acid (when X is OH) or appropriate acid derivative (whenX is an appropriate leaving group) to provide the compound of Formula 7.

8. Caspase-1 Inhibitor Having Formula 8, 8.1, Or 8.2

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2009/0281128 and US2004/0072850 (herewith incorporated byreference in their entireties). Preferred compounds described inpublished US2009/0281128 and US2004/0072850 for use in the methods ofthe present invention are referred to herein as caspase-1 inhibitorhaving Formula 8:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein X is N; Y is halo,trifluorophenoxy, or tetrafluorophenoxy; R² is C₁₋₆ straight chained orbranched alkyl; R³ is hydrogen, halo, OCF₃, CN, or CF₃; and R⁴ ishydrogen, halo, OCF₃, SR, CN, CF₃, Ar, or T-Ar; wherein: T is O or S; Ris a C₁₋₆ straight chained or branched alkyl; Ar is a phenyl ringoptionally substituted with 1-3 groups selected from halo, CH₃, CF₃, CN,OMe, OCF₃, and NR⁵R⁶; and R⁵ and R⁶ each is independently H or C₁₋₆straight chained or branched alkyl, or R⁵ and R⁶, taken together, form a5-7 membered ring optionally containing up to 3 heteroatoms selectedfrom O, S, NH, and N(C₁₋₆-straight chained or branched alkyl); providedthat when Y is halo, then both, R³ and R⁴, are not simultaneouslyhydrogen;a compound of Formula 8.1 (referred to herein as caspase-1 inhibitorhaving Formula 8.1):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: X is N; R² is ethyl,n-propyl, or isopropyl; R³ and R⁴ are each independently hydrogen, halo,OCF₃, CN, or CF₃; and Ar² is trifluorophenyl or tetrafluorophenyl;or a compound of Formula 8.2 (referred to herein as caspase-1 inhibitorhaving Formula 8.2):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: R² is ethyl, n-propyl, orisopropyl; R³ is hydrogen, halo, OCF₃, CN, or CF₃; R⁴ is halo, OCF₃, CN,CF₃, SR, or T-Ar; T is O or S; R is a C₁₋₆ straight chained or branchedalkyl; Ar is a phenyl ring optionally substituted with 1-3 groupsselected from halo, CH₃, CF₃, CN, OMe, OCF₃, and NR⁵R⁶; and R⁵ and R⁶each is independently H or C₁₋₆ straight chained or branched alkyl, orR⁵ and R⁶, taken together, form a 5-7 membered ring optionallycontaining up to 3 heteroatoms selected from O, S, NH, andN(C₁₋₆-straight chained or branched alkyl),

9. Caspase-1 Inhibitor Having Formula 9 Or 9.1

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2009/0093416 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2009/0093416 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 9:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: R¹ is R⁶C(O)—, HC(O)—,R⁶SO₂—, R⁶OC(O)—, (R⁶)₂NC(O)—, (R⁶)(H)NC(O)—, R⁶C(O)C(O)—, R⁶—,(R⁶)₂NC(O)C(O)—, (R⁶)(H)NC(O)C(O)—, or R⁶OC(O)C(O)—; R² is hydrogen,—CF₃, -halo, —OR⁷, —NO₂, —OCF₃, —CN, or R⁸; R³ is hydrogen or(C1-C4)-aliphatic-; R⁴ is —COOH or —COOR⁸; R⁵ is —CH₂F or—CH₂O-2,3,5,6-tetrafluorophenyl; R⁶ is(C1-C12)-aliphatic-(C3-C10)-cycloaliphatic-, (C6-C10)-aryl-,(C3-C10)-heterocyclyl-, (C5-C10)-heteroaryl-,(C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-,(C6-C10)-aryl-(C1-C12)-aliphatic-,(C3-C10)-heterocyclyl-(C1-C12)-aliphatic-,(C5-C10)-heteroaryl(C1-C12)-aliphatic-, or two R⁶ groups bound to thesame atom form together with that atom a 3- to 10-membered aromatic ornonaromatic ring; wherein any ring is optionally fused to a(C6-C10)aryl, (C5-C10)heteroaryl, (C3-C10)cycloalkyl, or a(C3-C10)heterocyclyl; wherein up to 3 aliphatic carbon atoms may bereplaced by a group selected from O, N, N(R), S, SO, and SO₂; andwherein R⁶ is substituted with up to 6 substituents independentlyselected from R; R is halogen, —OR⁷, —OC(O)N(R⁷)₂, —NO₂, —CN, —CF₃,—OCF₃, —R⁷, oxo, thioxo, ═NR⁷, ═N(OR⁷), 1,2-methylenedioxy,1,2-ethylenedioxy, —N(R⁷)₂, —SR⁷, —SOR⁷, —SO₂R⁷, —SO₂N(R⁷)₂, —SO₃R⁷,—C(O)R⁷, —C(O)C(O)R⁷, —C(O)C(O)OR⁷, —C(O)C(O)N(R⁷)₂, —C(O)CH₂C(O)R⁷,—C(S)R⁷, —C(S)OR⁷, —C(O)OR⁷, —OC(O)R⁷, —C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,—C(S)N(R⁷)₂, —(CH₂)₀₋₂NHC(O)R⁷, —N(R⁷)N(R⁷)COR⁷, —N(R⁷)N(R⁷)C(O)OR⁷,—N(R⁷)N(R⁷)CON(R⁷ ₂, —N(R⁷)SO₂R⁷, —N(R⁷)SO₂N(R⁷)₂, —N(R⁷)C(O)OR⁷,—N(R⁷)C(O)R⁷, —N(R⁷)C(S)R⁷, —N(R⁷)C(O)N(R⁷)₂, —N(R⁷)C(S)N(R⁷)₂,—N(COR⁷)COR⁷, —N(OR⁷)R⁷, —C(═NH)N(R⁷)₂, —C(O)N(OR⁷)R⁷, —C(═NOR⁷)R⁷,—OP(O)(OR⁷)₂, —P(O)(R⁷)₂, —P(O)(OR⁷)₂, or —P(O)(H)(OR⁷); two R⁷ groupstogether with the atoms to which they are bound form a 3- to 10-memberedaromatic or non-aromatic ring having up to 3 heteroatoms independentlyselected from N,N(R), O, S, SO, or SO₂, wherein the ring is optionallyfused to a (C6-C10)aryl, (C5-C10)heteroaryl, (C3-C10)cycloalkyl, or a(C3-C10)heterocyclyl, and wherein any ring has up to 3 substituentsselected independently from J₂; or each R⁷ is independently selectedfrom: hydrogen-, (C1-C12)-aliphatic-, (C3-C10)-cycloaliphatic-,(C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-, (C6-C10)-aryl-,(C6-C10)-aryl-(C1-C12)aliphatic-, (C3-C10)-heterocyclyl-,(C6-C10)-heterocyclyl-(C1-C12)aliphatic-, (C5-C10)-heteroaryl-, or(C5-C10)-heteroaryl-(C1-C12)-aliphatic-; wherein R⁷ has up to 3substituents selected independently from J₂; and J₂ is halogen, —OR⁷,—OC(O)N(R⁷)₂, —NO₂, —CN, —CF₃, —OCF₃, —R⁷, oxo, thioxo, ═N(R⁷), ═NO(R⁷),1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁷)₂, —SR⁷, —SOR⁷, —SO₂R⁷,—SO₂N(R⁷)₂, —SO₃R⁷, —C(O)R⁷, —C(O)C(O)R⁷, —C(O)C(O)OR⁷, —C(O)C(O)N(R⁷)₂,—C(O)CH₂C(O)R⁷, —C(S)R⁷, —C(S)OR⁷, —C(O)R⁷, —OC(O)R⁷, —C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —C(S)N(R⁷)₂, —(CH₂)₀₋₂ NHC(O)R⁷, —N(R⁷)N(R⁷)COR⁷,—N(R⁷)N(R⁷)C(O)OR⁷, —N(R⁷)N(R⁷)CON(R⁷)₂, —N(R⁷)SO₂R⁷—N(R⁷)SO₂N(R⁷)₂,—N(R⁷)C(O)OR⁷, —N(R⁷)C(O)R⁷, —N(R⁷)C(S)R⁷—N(R⁷)C(O)N(R⁷)₂,—N(R⁷)C(S)N(R⁷)₂, —N(COR⁷)COR⁷, —N(OR⁷)R⁷, —CN, —C(═NH)N(R⁷)₂,—C(O)N(OR⁷)R⁷, —C(═NOR⁷)R⁷, —OP(O)(OR⁷)₂, —P(O)(R⁷)₂, —P(O)(OR⁷)₂, or—P(O)(H)(OR⁷); and R⁸ is (C1-C12)-aliphatic-(C3-C10)-cycloaliphatic-,(C6-C10)-aryl-, (C3-C10)-heterocyclyl-, (C5-C10)-heteroaryl-,(C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-,(C6-C10)-aryl-(C1-C12)-aliphatic-,(C3-C10)-heterocyclyl-(C1-C12)-aliphatic-, or(C5-C10)-heteroaryl(C1-C12)-aliphatic-, wherein up to 3 aliphatic carbonatoms may be replaced with a group selected from O, N,N(R), S, SO, andSO₂; and wherein R⁸ is optionally substituted with up to 6 substituentsindependently selected from R;or a compound of Formula 9.1 (referred to herein as caspase-1 inhibitorhaving Formula 9.1):

wherein: R¹ is R⁶C(O)—, R⁶SO₂—, R⁶OC(O)—, (R⁶)₂NC(O)—, R⁶C(O)C(O)—, R⁶—,(R⁶)₂NC(O)C(O)—, or R⁶OC(O)C(O)—; R² is hydrogen, —CF₃, -halo, —OR⁷,—NO₂, —OCF₃, —CN, or R⁸; R³ is hydrogen or (C1-C4)-aliphatic-; R⁴ is—COOH or —COOR⁸; R⁵ is —CH₂F or —CH₂O-2,3,5,6-tetrafluorophenyl; R⁶ is(C1-C12)-aliphatic-(C3-C10)-cycloaliphatic-, (C6-C10)-aryl-,(C3-C10)-heterocyclyl-, (C5-C10)-heteroaryl-,(C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-,(C6-C10)-aryl-(C1-C12)-aliphatic-,(C3-C10)-heterocyclyl-(C1-C12)-aliphatic-,(C5-C10)-heteroaryl(C1-C12)-aliphatic-, or two R⁶ groups bound to thesame atom form together with that atom a 3- to 10-membered aromatic ornonaromatic ring; wherein any ring is optionally fused to a(C6-C10)aryl, (C5-C10)heteroaryl, (C3-C10)cycloalkyl, or a(C3-C10)heterocyclyl; wherein up to 3 aliphatic carbon atoms may bereplaced by a group selected from O, N, N(R), S, SO, and SO₂; andwherein R⁶ is substituted with up to 6 substituents independentlyselected from R; R is halogen, —OR⁷, —OC(O)N(R⁷)₂, —NO₂, —CN, —CF₃,—OCF₃, —R⁷, oxo, thioxo, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁷)₂,—SR⁷, —SOR⁷, —SO₂R⁷, —SO₂N(R⁷)₂, —SO₃R⁷, —C(O)R⁷, —C(O)C(O)R⁷,—C(O)CH₂C(O)R⁷, —C(S)R⁷, —C(O)OR⁷, —OC(O)R⁷, —C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,—C(S)N(R⁷)₂, —(CH₂)₀₋₂NHC(O)R⁷,—N(R⁷)N(R⁷)COR⁷,—N(R⁷)N(R⁷)C(O)R⁷—N(R⁷)N(R⁷)CON(R⁷)₂—, —N(R⁷)SO₂R⁷,—N(R⁷)SO₂N(R⁷)₂, —N(R⁷)C(O)OR⁷, —N(R⁷)C(O)R⁷,—N(R⁷)C(S)R⁷—N(R⁷)C(O)N(R⁷)₂, —N(R⁷)C(S)N(R⁷)₂, —N(COR⁷)COR⁷, —N(OR⁷)R⁷,—C(═NH)N(R⁷)₂, —C(O)N(OR⁷)R⁷, —C(═NOR⁷)R⁷, —OP(O)(OR⁷)₂, —P(O)(R⁷)₂,—P(O)(OR⁷)₂, or —P(O)(H)(OR⁷); two R⁷ groups together with the atoms towhich they are bound form a 3- to 10-membered aromatic or non-aromaticring having up to 3 heteroatoms independently selected from N,N(R), O,S, SO, or SO₂, wherein the ring is optionally fused to a (C6-C10)aryl,(C5-C10)heteroaryl, (C3-C10)cycloalkyl, or a (C3-C10)heterocyclyl, andwherein any ring has up to 3 substituents selected independently fromJ₂; or each R⁷ is independently selected from: hydrogen-,(C1-C12)-aliphatic-, (C3-C10)-cycloaliphatic-,(C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-, (C6-C10)-aryl-,(C6-C10)-aryl-(C1-C12)aliphatic-, (C3-C10)-heterocyclyl-,(C6-C10)-heterocyclyl-(C1-C12)aliphatic-, (C5-C10)-heteroaryl-, or(C5-C10)-heteroaryl-(C1-C12)-aliphatic-; wherein R⁷ has up to 3substituents selected independently from J₂; and J₂ is halogen, —OR⁷,—OC(O)N(R⁷)₂, —NO₂, —CN, —CF₃, —OCF₃, —R⁷, oxo, thioxo,1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁷)₂, —SR⁷, —SOR⁷, —SO₂R⁷,—SO₂N(R⁷)₂, —SO₃R⁷, —C(O)R⁷, —C(O)C(O)R⁷, —C(O)CH₂C(O)R⁷, —C(S)R⁷,—C(O)OR⁷, —OC(O)R⁷, —C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —C(S)N(R⁷)₂,—(CH₂)₀₋₂NHC(O)R⁷, —N(R⁷)N(R⁷)COR⁷, —N(R⁷)N(R⁷)C(O)OR⁷,—N(R⁷)N(R⁷)CON(R⁷)₂, —N(R⁷)SO₂R⁷, —N(R⁷)SO₂N(R⁷)₂,—N(R⁷)C(O)OR⁷7-N(R⁷)C(O)R⁷, —N(R⁷)C(S)R⁷, —N(R⁷)C(O)N(R⁷)₂,—N(R⁷)C(S)N(R)₂, —N(COR⁷)COR⁷, —N(OR⁷)R⁷, —CN, —C(═NH)N(R⁷)₂,—C(O)N(OR⁷)R⁷—C(═NOR⁷)R⁷, —OP(O)(OR⁷)₂, —P(O)(R⁷)₂, —P(O)(OR⁷)₂, or—P(O)(H)(OR⁷); and R⁸ is (C1-C12)-aliphatic-(C3-C10)-cycloaliphatic-,(C6-C10)-aryl-, (C3-C10)-heterocyclyl-, (C5-C10)-heteroaryl-,(C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-,(C6-C10)-aryl-(C1-C12)-aliphatic-,(C3-C10)-heterocyclyl-(C1-C12)-aliphatic-, or(C5-C10)-heteroaryl(C1-C12)-aliphatic-, wherein up to 3 aliphatic carbonatoms may be replaced with a group selected from O, N,N(R), S, SO, andSO₂.

10. Caspase-1 Inhibitor Having Formula 10

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2008/0286201 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2008/0286201 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 10:

Z¹—(X¹)_(m1)-Asp(R¹)-Xaa1-Xaa2-Asp(R²)-(A)_(n)-[IM]  Formula 10

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: Z¹ is attached to theN-terminus of X¹ or the Asp residue, and is H or a metabolism inhibitinggroup; X¹ is a cell membrane permeable leader sequence peptide of 4 to20 amino acids which facilitates cell membrane transport from theoutside to the inside of a mammalian cell in vivo; Xaa1 is Glu(R³) orMet; Xaa2 is Val or is Gln when Xaa1 is Met; Asp is aspartic acid;-(A)_(n)- is a linker group wherein each A is independently —CR₂—,—CR═CR—, —C═C—, —CR₂CO₂—, —CO₂CR₂—, —NRCO—, —CONR—, —NR(C═O)NR—,—NR(C═S)NR—, —SO₂NR—, —NRSO₂—, —CR₂OCR₂—, —CR₂SCR₂—, —CR₂NRCR₂—, a C₄₋₈cycloheteroalkylene group, a C₄₋₈ cycloalkylene group, a C₅₋₁₂ arylenegroup, or a C₃₋₁₂ heteroarylene group, an amino acid, a sugar or amonodisperse polyethyleneglycol (PEG) building block; each R isindependently chosen from H, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl,C₁₋₄ alkoxyalkyl or C₁₋₄ hydroxyalkyl; R¹, R² and R³ are independentlyR′ groups which are attached at the carboxy side chain of the Asp or Gluamino acid residue, where each R′ is chosen from H, C₁₋₈ alkyl, C₂₋₈alkoxyalkyl, C₅₋₁₂ aryl or C₅₋₁₆ aralkyl; m₁ is 0 or 1; n is an integerof value 0 to 10; IM is an optional imaging moiety which comprises agamma-emitting radioactive halogen or a positron-emitting radioactivenon-metal.

11. Caspase-1 Inhibitor Having Formula 11

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2007/0010457 and US2003/0096737 (herewith incorporated byreference in their entireties). Preferred compounds described inpublished US2007/0010457 and US2003/0096737 for use in the methods ofthe present invention are referred to herein as caspase-1 inhibitorhaving Formula 11:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: R¹ is hydrogen, CN, CHN₂,R, or —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group, or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving group,—OR, —SR, —OC═O(R), or —OPO(R³)(R⁴); R³ and R⁴ are independently R orOR; R² is CO₂H, CH₂CO₂H, or optionally substituted esters, amides orisosteres thereof; A is C═O or SO₂; X¹ is oxygen, sulfur, —NH, or —CH₂,wherein —NH is optionally substituted by an alkyl group, a cycloalkylgroup, a (cycloalkyl)alkyl group, an amino acid N-terminal protectinggroup, or COR and —CH₂ is optionally substituted by fluorine, an alkylgroup, a cycloalkyl group, a (cycloalkyl)alkyl group, an aralkyl group,an aryl group, an alkyloxy group, an alkylthioxy group, an aryloxygroup, an arylthioxy group, an oxo group (i.e., =0), or a NHCOR group;X² is oxygen, sulfur, —NH, or —CH₂, wherein —NH is optionallysubstituted by an alkyl group, or an amino acid N-terminal protectinggroup and —CH₂ is optionally substituted by an alkyl group, an arylgroup, an alkyloxy group, an alkylthioxy group, an aryloxy group, anarylthioxy group, or an oxo (i.e., ═O) group, a NHCOR group; X¹ and X²optionally form part of a phenyl ring that is fused to the adjoiningring Q; X³ is CH₂ or X² and X³ optionally form part of a phenyl ringthat is fused to the adjoining ring Q, provided that when X² forms aring with X³, then X² does not form a ring with X¹; any two hydrogensattached to adjacent positions in ring Q are optionally replaced by adouble bond; and Z is an optionally substituted ring selected from thegroup consisting of a carbocyclic, an aryl, a saturated heterocycle, apartially saturated heterocycle, and a heteroaryl wherein the ring isconnected to A at a ring carbon.

12. Caspase-1 Inhibitor Having Formula 12

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2006/0160862 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2006/0160862 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 12:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: R¹ is R⁶C(O)—, HC(O)—,R⁶SO₂—, R⁶OC(O)—, (R⁶)₂NC(O)—, (R⁶)(H)NC(O)—, R⁶C(O)C(O)—,(R⁶)₂NC(O)C(O)—, (R⁶)(H)NC(O)C(O)—, or R⁶OC(O)C(O)—; R² is hydrogen,—CF₃, halo, —OR⁷, NO₂, —OCF₃, —CN, or R⁸; R³ is -T-R⁹; R⁴ is —COOH or—COOR⁸; R⁵ is —CH₂F or —CH₂O-2,3,5,6-tetrafluorophenyl; R⁶ is R^(6a) orR^(6b); two R⁶ groups, together with the same atom to which they arebound, optionally form a 3- to 10-membered aromatic or nonaromatic ring;wherein the ring is optionally fused to a (C6-C10)aryl,(C5-C10)heteroaryl, (C3-C10)cycloalkyl, or a (C3-C10)heterocyclyl;wherein up to 3 aliphatic carbon atoms may be replaced by a groupselected from O, N, N(R⁷), S, SO, and SO₂; and wherein each R⁶ isindependently substituted with up to 6 substituents independentlyselected from R; R^(6a) and R^(6b) are each independently(C1-C3)-aliphatic-, (C4-C12)-aliphatic-, (C3-C10)-cycloaliphatic-,(C6-C10)-aryl-, (C3-C10)-heterocyclyl-, (C5-C10)-heteroaryl-,(C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-,(C6-C10)-aryl-(C1-C12)-aliphatic-,(C3-C10)-heterocyclyl-(C1-C12)-aliphatic-,(C5-C10)-heteroaryl(C1-C12)-aliphatic-; R is halogen, —OR⁷, OC(O)N(R⁷)₂,NO₂, CN, CF₃, OCF₃, R⁷, oxo, thioxo, ═NR⁷, ═N(OR⁷), 1,2-methylenedioxy,1,2-ethylenedioxy, —N(R⁷)₂, —SR⁷, —SOR⁷, —SO₂R⁷, —SO₂N(R⁷)₂, —SO₃R⁷,—C(O)R⁷, —C(O)C(O)R⁷, —C(O)C(O)OR⁷, —C(O)C(O)N(R⁷)₂, —C(O)CH₂C(O)R⁷,—C(S)R⁷, —C(S)OR⁷, —C(O)OR⁷, —OC(O)R⁷, —C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,—C(S)N(R⁷)₂, —(CH₂)₀₋₂NHC(O)R⁷, —N(R⁷)N(R⁷)COR⁷, —N(R⁷)N(R⁷)C(O)OR⁷,—N(R⁷)N(R⁷)CON(R⁷)₂, —N(R⁷)SO₂R⁷, —N(R⁷)SO₂N(R⁷)₂, —N(R⁷)C(O)OR⁷,—N(R⁷)C(O)R⁷, —N(R⁷)C(S)R⁷, —N(R⁷)C(O)N(R⁷)₂, —N(R⁷)C(S)N(R⁷)₂,—N(COR⁷)COR⁷, —N(OR⁷)R⁷, —C(═NR⁷)N(R⁷)₂, —C(O)N(OR⁷)R⁷, —C(═NOR⁷)R⁷,—OP(O)(OR⁷)₂, —P(O)(R⁷)₂, —P(O)(OR⁷)₂, or —P(O)(H)(OR⁷); two R⁷ groupstogether with the atoms to which they are bound optionally form a 3- to10-membered aromatic or non-aromatic ring having up to 3 heteroatomsindependently selected from N, N(R⁷), O, S, SO, or SO₂, wherein the ringis optionally fused to a (C6-C10)aryl, (C5-C10)heteroaryl,(C3-C10)cycloalkyl, or a (C3-C10)heterocyclyl, and wherein any ring hasup to 3 substituents selected independently from J₂; or each R⁷ isindependently selected from: hydrogen-, (C1-C12)-aliphatic-,(C3-C10)-cycloaliphatic-, (C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-,(C6-C10)-aryl-, (C6-C10)-aryl-(C1-C12)aliphatic-,(C3-C10)-heterocyclyl-, (C6-C10)-heterocyclyl-(C1-C12)aliphatic-,(C5-C10)-heteroaryl-, or (C5-C10)-heteroaryl-(C1-C12)-aliphatic-;wherein R⁷ has up to 3 substituents selected independently from J₂; andJ₂ is halogen, —OR′, —OC(O)N(R⁷)₂, —NO₂, —CN, —CF₃, —OCF₃, —R⁷, oxo,thioxo, ═NR⁷, ═NO(R⁷), 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁷)₂,—SR⁷, —SOR⁷, —SO₂R⁷, —SO₂N(R⁷)₂, —SO₃R⁷, —C(O)R⁷, —C(O)C(O)R⁷,—C(O)C(O)OR⁷, —C(O)C(O)N(R⁷)₂, —C(O)CH₂C(O)R⁷, —C(S)R⁷, —C(S)OR⁷,—C(O)OR⁷, —OC(O)R⁷, —C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —C(S)N(R⁷)₂,—(CH₂)₀₋₂NHC(O)R⁷, —N(R⁷)N(R⁷)COR⁷, —N(R⁷)N(R⁷)C(O)OR⁷,—N(R⁷)N(R⁷)CON(R⁷)₂, —N(R⁷)SO₂R⁷, —N(R⁷)SO₂N(R⁷)₂, —N(R⁷)C(O)OR⁷,—N(R⁷)C(O)R⁷, —N(R⁷)C(S)R⁷, —N(R⁷)C(O)N(R⁷)₂, —N(R⁷)C(S)N(R⁷)₂,—N(COR⁷)COR₇, —N(OR⁷)R⁷, —CN, —C(═NR⁷)N(R⁷)₂, —C(O)N(OR⁷)R⁷,—C(═NOR⁷)R⁷, —OP(O)(OR⁷)₂, —P(O)(R⁷)₂, —P(O)(OR⁷)₂, or —P(O)(H)(OR⁷);and R⁸ is (C1-C12)-aliphatic-, (C3-C10)-cycloaliphatic-, (C6-C10)-aryl-,(C3-C10)-heterocyclyl-, (C5-C10)-heteroaryl-,(C3-C10)-cycloaliphatic-(C1-C12)-aliphatic-,(C6-C10)-aryl-(C1-C12)-aliphatic-,(C3-C10)-heterocyclyl-(C1-C12)-aliphatic-, or(C5-C10)-heteroaryl-(C1-C12)-aliphatic-, wherein up to 3 aliphaticcarbon atoms may be replaced with a group selected from O, N, N(R⁷), S,SO, and SO₂; and wherein R⁸ is optionally substituted with up to 6substituents independently selected from R; T is a direct bond or(C1-C6) aliphatic wherein up to 2 aliphatic carbon atoms in T may beoptionally replaced with S, —SO—, SO₂, O, N(R⁷), or N in a chemicallystable arrangement; wherein each T may be optionally substituted with upto 3 R substituents; R⁹ is optionally substituted (C6-C10)-aryl or(C5-C10)-heteroaryl.

13. Caspase-1 Inhibitor Having Formula 13

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2004/0116355 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2004/0116355 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 13:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein: R₁ is an optionallysubstituted alkyl or hydrogen; R₂ is hydrogen or optionally substitutedalkyl; R₃ is an alkyl, saturated carbocyclic, partially saturatedcarbocyclic, aryl, saturated heterocyclic, partially saturatedheterocyclic or heteroaryl group, wherein said group is optionallysubstituted; X is O, S, NR₄ or (CR₄R₅)_(n), where R₄ and R₅ are, at eachoccurrence, independently selected from the group consisting ofhydrogen, alkyl and cycloalkyl, and n is 0, 1, 2 or 3; or X is NR₄, andR₃ and R₄ are taken together with the nitrogen atom to which they areattached to form a saturated heterocyclic, partially saturatedheterocyclic or heteroaryl group, wherein said group is optionallysubstituted; or X is CR₄R₅, and R₃ and R₄ are taken together with thecarbon atom to which they are attached to form a saturated carbocyclic,partially saturated carbocyclic, aryl, saturated heterocyclic, partiallysaturated heterocyclic or oxygen-containing heteroaryl group, whereinsaid group is optionally substituted; and Y is a residue of a natural ornon-natural amino acid; provided that when X is O, then R₃ is notunsubstituted benzyl or t-butyl; and when X is CH₂, then R₃ is nothydrogen.

14. Caspase-1 Inhibitor Having Formula 14

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2004/0048895 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2004/0048895 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 14:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R₁ is chosen fromoptionally substituted aryl, optionally substituted aralkyl, optionallysubstituted heteroaryl, and optionally substituted heteroaralkyl; L is alinker; R₂ is chosen from optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl, and optionallysubstituted heteroaralkyl; and a compound of Formula 14, wherein Y is

15. Caspase-1 Inhibitor Having Formula 15

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2004/0019017 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2004/0019017 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 15:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is a saturated orunsaturated, straight-chain or branched, substituted or unsubstitutedhydrocarbon chain; R² is H or a phospholipid head group; X is a directcovalent bond or a group C(O)LR³; wherein L is a saturated orunsaturated, straight-chain or branched, substituted or unsubstitutedhydrocarbon chain having from 2 to 15 carbon atoms, which optionallyincludes cyclic elements, and is optionally interrupted by one or moreatoms selected from the group consisting of oxygen, sulfur and N(R⁴), R³is selected from the group consisting of O, S and N(R⁴); wherein R⁴ is asaturated or unsaturated hydrocarbon chain having 1 to 6 carbon atoms;and Y is a residue of a caspase-1 inhibitor.

16. Caspase-1 Inhibitor Having Formula 16, 16.1, 16.2, 16.3, 16.4, 16.5,16.6, Or 16.7

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2003/0232846 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2003/0232846 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 16:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, or —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving groupor —OR, —SR, —OC═O(R), or —OPO(R⁸) (R⁹); R⁸ and R⁹ are independentlyselected from R or OR; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is hydrogen or a C₁₋₆ straight chained or branchedalkyl; Ring A contains zero to two double bonds, and is optionally fusedto a saturated or unsaturated five to seven membered ring containingzero to three heteroatoms; X₁ and X₃ in Ring A are independentlyselected from nitrogen or carbon, and X₂ is selected from a valencebond, oxygen, sulfur, nitrogen or carbon, wherein any X with suitablevalence may bear a substituent; each carbon with suitable valence inRing A, including the fused ring if present, is independentlysubstituted by hydrogen, halo, R, OR, SR, OH, NO₂, CN, NH₂, NHR, N(R)₂,NHCOR, NHCONHR, NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR,CON(R)₂, S(O)₂R, SONH₂, S(O)R, SO₂NHR, NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂,═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or ═NR; each substitutable nitrogenin Ring A is substituted by hydrogen, R, COR, S(O)₂R, or CO₂R; providedthat when X₃ is a carbon, a substituent on X₃ is attached by an atomother than nitrogen; and further provided that at least one X in Ring Ais a nitrogen;a compound having Formula 16.1 (referred to herein as caspase-1inhibitor having Formula 16.1):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving group,—OR, —SR, —OC═O(R), or —OPO(R⁸) (R⁹); R⁸ and R⁹ are each independentlyselected from R or OR; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is hydrogen or a C₁₋₆ straight chained or branchedalkyl; each of R⁴-R⁶ is independently selected from hydrogen, halo, R,OR, SR, aryl, substituted aryl, OH, NO₂, CN, NH₂, NHR, N(R)₂, NHCOR,NHCONHR, NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR, CON(R)₂,S(O)₂R, SONH₂, S(O)R, SO₂NHR, or NHS(O)₂R; and R⁷ is selected fromhydrogen, halo, R, OR, SR, aryl, substituted aryl, OH, CN, CO₂R, CO₂H,COR, CONHR, CON(R)₂, S(O)₂R, SONH₂, S(O)R, or SO₂NHR;a compound having Formula 16.2 (referred to herein as caspase-1inhibitor having Formula 16.2):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, or —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving groupor —OR, —SR, —OC═O(R), or —OPO(R⁸) (R⁹); R⁸ and R⁹ are eachindependently selected from R or OR; R² is CO₂H, CH₂CO₂H, or esters,amides or isosteres thereof; R³ is hydrogen or a C₁₋₆ straight chainedor branched alkyl; R⁶ is selected from hydrogen, halo, R, OR, SR, aryl,substituted aryl, OH, NO₂, CN, NH₂, NHR, N(R)₂, NHCOR, NHCONHR,NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR, CON(R)₂, S(O)₂R,SONH₂, S(O)R, SO₂NHR, or NHS(O)₂R; and R⁷ is selected from hydrogen,halo, R, OR, SR, aryl, substituted aryl, OH, CN, CO₂R, CO₂H, COR, CONHR,CON(R)₂, S(O)₂R, SONH₂, S(O)R, or SO₂NHR;a compound having Formula 16.3 (referred to herein as caspase-1inhibitor having Formula 16.3):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, or —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving groupor —OR, —SR; —OC═O(R), or —OPO(R⁸) (R⁹); R⁸ and R⁹ are independentlyselected from R or OR; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is hydrogen or a C₁₋₆ straight chained or branchedalkyl; R⁴ and R⁵ are each independently selected from hydrogen, halo, R,OR, SR, aryl, substituted aryl, OH, NO₂, CN, NH₂, NHR, N(R)₂, NHCOR,NHCONHR, NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR, CON(R)₂,S(O)₂R, SONH₂, S(O)R, SO₂NHR, NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂, ═N—OR,═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or ═NR;a compound having Formula 16.4 (referred to herein as caspase-1inhibitor having Formula 16.4):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving groupor —OR, —SR, —OC═O(R), or —OPO(R⁸)(R⁹); R⁸ and R⁹ are independentlyselected from R or OR; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is hydrogen or a C₁₋₆ straight chained or branchedalkyl; R⁴ is independently selected from hydrogen, halo, R, OR, SR,aryl, substituted aryl, OH, NO₂, CN, NH₂, NHR, N(R)₂, NHCOR, NHCONHR,NHCON(R) 2, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR₂, CON(R)₂, S(O)₂R,SONH₂, S(O)R, SO₂NHR, or NHS(O)₂R; R⁷ is selected from hydrogen, halo,R, OR, SR, aryl, substituted aryl, OH, CN, CO₂R, CO₂H, COR, CONHR,CON(R)₂, S(O)₂R, SONH₂, S(O)R, or SO₂NHR;a compound having Formula 16.5 (referred to herein as caspase-1inhibitor having Formula 16.5):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving groupor —OR, —SR, —OC═O(R), or —OPO(R⁸) (R⁹); R⁸ and R⁹ are independentlyselected from R or OR; R² is CO₂H, CH₂CO₂H, or esters or isosteresthereof; R³ is hydrogen or a C₁₋₆ straight chained or branched alkyl; R⁴and R⁵ are each independently selected from hydrogen, halo, R, OR, SR,aryl, substituted aryl, OH, NO₂, CN, NH₂, NHR, N(R)₂, NHCOR, NHCONHR,NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR, CON(R)₂, S(O)₂R,SONH₂, S(O)R, SO₂NHR, or NHS(O)₂R; and the fused ring is an aromatic ornon-aromatic heterocyclic ring;a compound having Formula 16.6 (referred to herein as caspase-1inhibitor having Formula 16.6):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, or —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving groupor —OR, —SR, —OC═O(R), or —OPO(R⁸) (R⁹); R⁸ and R⁹ are independentlyselected from R or OR; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is hydrogen or a C₁₋₆ straight chained or branchedalkyl; and R⁴ is independently selected from hydrogen, halo, R, OR, SR,aryl, substituted aryl, OH, NO₂, CN, NH₂, NHR, N(R)₂, NHCOR, NHCONHR,NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR, CON(R)₂, S(O)₂R,SONH₂, S(O)R, SO₂NHR, or NHS(O)₂R;or a compound having Formula 16.7 (referred to herein as caspase-1inhibitor having Formula 16.7):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, or —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving groupor —OR, —SR, —OC═O(R), or —OPO(R⁸) (R⁹); R⁸ and R⁹ are independentlyselected from R or OR; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is hydrogen or a C₁₋₆ straight chained or branchedalkyl; each of R⁴ and R⁶ is independently selected from hydrogen, halo,R, OR, SR, aryl, substituted aryl, OH, NO₂, CN, NH₂, NHR, N(R)₂, NHCOR,NHCONHR, NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR, CON(R)₂,S(O)₂R, SONH₂, S(O)R, SO₂NHR, or NHS(O)₂R; and R⁷ is selected fromhydrogen, halo, R, OR, SR, aryl, substituted aryl, OH, CN, CO₂R, CO₂H,COR, CONHR, CON(R)₂, S(O)₂R, SONH₂, S(O)R, or SO₂NHR.

17. Caspase-1 Inhibitor Having Formula 17, 17.1, 17.2, 17.3, 17.4, 17.5,17.6, 17.7, 17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15, 17.16,17.17, 417.18, 17.19, 17.20. 17.21, and 17.22

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2010/0040607 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2010/0040607 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 17:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein Ring A is an optionallysubstituted piperidine, tetrahydroquinoline or tetrahydroisoquinolinering, R¹ is hydrogen, CN, CHN₂, R, or CH₂Y, R is an optionallysubstituted group selected from an aliphatic group, an aryl group, or anaralkyl group, Y is an electronegative leaving group, R² is CO₂H,CH₂CO₂H, or esters, amides or isosteres thereof, R³ is hydrogen, anoptionally substituted aryl group, an optionally substituted aralkylgroup, or an optionally substituted C₁₋₆ aliphatic group, R⁴ is anoptionally substituted group selected from an aryl group or aheterocyclyl group, or R³ and R⁴ taken together with the nitrogen towhich they are attached optionally form is a substituted orunsubstituted monocyclic, bicyclic or, tricyclic ring;a compound having Formula 17.1 (referred to herein as caspase-1inhibitor having Formula 17.1):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is H, an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl, or heteroalkylheteroaryl moiety; n is 0 or 1; A isCR^(A), C(R^(A))₂, C═O, S, NR^(A), N(R^(A))₂, or O; B is CR^(B),C(R^(B))₂, C═O, S, NR^(B), N(R^(B))₂, or O; D is CR^(D), C(R^(D))₂, C═O,S, NR^(D), N(R^(D))₂, or O; E is CR^(E), C(R^(E))₂, C═O, S, NR^(E),N(R^(E))₂, or O; G is CR^(G), C(R^(G))₂, C═O, S, NR^(G), N(R^(G))₂, orO; J is CR^(J); each of A-B, B-D, D-E, E-G, G-J and A-J are connected bya single or double bond as valency and stability permits; eachoccurrence of R^(A), R^(B), R^(D), R^(E), R^(G) and R^(J) isindependently hydrogen, halogen, —OR², —N (R²)₂, —SR², —CN, —COOR²,—COR², —CON(R², —SOR², —SO₂R², —SO₂N(R²)₂, —NR² SO₂R², —O(C═O)N(R²)₂,—NR² (C═O)N(R²)₂, —NR² (C═S)N(R²)₂, —NR² SO₂N(R²)₂, or an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl, or heteroalkylheteroaryl moiety optionallyindependently substituted with one or more occurrences of R², whereineach occurrence of R² is independently hydrogen, halogen, —OR³, —N(R³)₂,—SR³, —CN, —COOR³, —COR³, —CON(R³)₂, —SOR³, —SO₂R³, —SO₂N(R³)₂,—NR³SO₂R³, —O(C═O)N(R³)₂, —NR³ (CO)N(R³)₂, —NR³ (C═S)N(R³)₂, —NR³SO₂N(R³2, or an aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl,alkylheteroaryl, heteroalkylaryl, or heteroalkylheteroaryl moiety;wherein each occurrence of R³ is independently hydrogen, or analiphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl,alkylheteroaryl, heteroalkylaryl, or heteroalkylheteroaryl moiety, andwherein at least one of R^(B) or R^(D) comprises —SR², —SOR², —SO₂R²,—SO₂N(R²)₂, —NR² SO₂R², —N(R²)₂, —(C═O)N(R²)₂, —NR²(C—O)R²,—O(C═O)N(R²)₂, —NR² (C═O)N(R²)₂, —NR² (C═S)N(R²)₂, —NR² SO₂N(R²)₂, or isan alkyl or heteroalkyl group substituted with one or more occurrencesof R², wherein R² is —SR³, —SOR³, —SO₂R³, —SO₂N(R³, —NR³ SO₂R³, —N(R³)₂,—C═O)N(R³)₂, —NR³ (C═O)R³, —O(C═O)N(R³)₂, —NR³ (C═O)N(R³)₂, —NR³(C═S)N(R³)₂, —NR³ SO₂N(R³)₂, wherein R³ is an aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl,heteroalkylaryl, or heteroalkylheteroaryl moiety, whereby each of theforegoing aliphatic, heteroaliphatic, alkyl and heteroalkyl moieties maybe independently substituted or unsubstituted, cyclic or acyclic, linearor branched, and each of the foregoing aryl, heteroaryl, alkylaryl,alkylheteroaryl, heteroalkylaryl and heteroalkylheteroaryl moieties maybe independently substituted or unsubstituted;a compound having Formula 17.2 (referred to herein as caspase-1inhibitor having Formula 17.2):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is selected from thegroup consisting of: OH, C₁₋₄ alkyl, HET, Aryl, C₁₋₆ alkoxy, NH₂, NHC₁₋₆alkyl, N(C₁₋₆ alkyl)₂, C₁₋₆ alkylC(O), C₁₋₆ alkylS(O)_(y),Aryl-S(O)_(y), HET-S(O)_(y) wherein y is 0, 1 or 2, Aryl-C(O) andHET-C(O), the alkyl and alkyl portions of which being optionallysubstituted with 1-2 members selected from the group consisting of: OH,Aryl¹, HET, halo, NH₂, NHCH₃, N(CH₃)₂, CO₂H, CF₃ and C₁₋₄-acyl; Arylrepresents a C₆₋₁₄ aromatic 1-3 ring system optionally substituted with1-3 members selected from OH, C₁₋₆ alkyl, OC₁₋₆alkyl, Aryl¹, HET, halo,NH₂, NHCH₃, N(CH₃)₂, CF₃, CO₂H and C₁₋₄ acyl; Aryl¹ represents a C₆₋₁₄membered aromatic ring system having 1-3 rings and optionallysubstituted with 1-3 members selected from the group consisting of: OH,HET, halo, NH₂, NHCH₃, N(CH₃)₂, CO₂H and C₁₋₄-acyl; HET represents a 5to 15 membered aromatic, partially aromatic or non-aromatic ring system,containing 1-4 heteroatoms selected from O, S and N, and optionallysubstituted with 1-2 oxo groups and 1-3 groups selected from halo, C₁₋₄alkyl, C₁₋₄alkoxy, CF₃ and C₁₋₄ acyl; R^(a) and R^(b) independentlyrepresent a member selected from the group consisting of: H, Aryl,C₁₋₆alkyl optionally substituted by 1-3 of halo, OR⁴, SR⁴ and C₅₋₇cycloalkyl optionally containing one heteroatom selected from O, S andNR⁵, or in the alternative, R^(a) and R^(b) are taken in combination andrepresent a non-aromatic carbocyclic 4-7 membered ring, optionallycontaining one heteroatom selected from O, S and NR⁵; R⁴ is selectedfrom the group consisting of: H, C₁₋₅ alkyl, Aryl and Aryl-CIA alkyloptionally substituted with 1-2 groups selected from halo and C₁₋₄alkyl; R⁵ is H, C₁₋₄ alkyl or C₁₋₄ acyl; R^(c) and R^(d) eachindependently represents a member selected from the group consisting of:H, C₁₋₆ alkyl and Aryl, or in the alternative, R^(c) and R^(d) are takenin combination and represent a non-aromatic carbocyclic ring of 3-7members, optionally containing one heteroatom selected from O, S andNR⁵; n is an integer from 0-6 inclusive; R² represents H, halo or C₁₋₆alkyl; R³ represents H, C₁₋₆ alkyl, Aryl, HET, C₁₋₆ alkylSR⁶, C₁₋₆alkylOR⁶, C₁₋₆ alkylOC(O)R⁷ or C₁₋₆alkylNR⁸R⁹; R⁶ represents C₁₋₆ alkyl,Aryl, HET or Aryl-C₁₋₆ alkyl, said alkyl and the alkyl portions beingoptionally substituted with 1-3 members selected from the groupconsisting of: OH, halo, NH₂, NHCH₃, N(CH₃)₂, CO₂H, CF₃ and C₁₋₄ acyl;R⁷ represents C₁₋₈ alkyl, Aryl or HET; R⁸ and R⁹ independently representH, C₁₋₁₀ alkyl, Aryl, HET, C₆₋₄ alkylN(C₁₋₆ alkyl)₀₋₂, Aryl-C₁₋₆ alkyl,C₁₋₆ alkylOH, or C₁₋₄ alkylOC₁₋₆ alkyl, or R⁸ and R⁹ are taken incombination with the nitrogen atom to which they are attached andrepresent a 3-10 membered ring system containing 1-4 heteroatomsselected from O, S, N and optionally substituted with 1-2 oxo groups,and 1-3 groups selected from C₁₋₄ alkyl, HET, CO₂R^(c) and C(O)N(R)₂,said alkyl and alkyl portions being optionally substituted with 1-3groups selected from halo, C₁₋₃ alkyl, hydroxyl C₁₋₃ alkyl, C₁₋₃ alkoxy,C₁₋₃ alkoxy C₁₋₃ alkyl and Aryl¹, and R¹⁰ represents H, C₁₋₂₀ alkyl,aryl or HU, with aryl and HET as previously described;a compound having Formula 17.3 (referred to herein as caspase-1inhibitor having Formula 17.3):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein W is a bond, —CH₂—; —C(O)—or —C(O)CH₂—; Z is selected from the group consisting of: (1) H, (2)C₁₋₁₁alkyl, (3) C₃₋₁₁cycloalkyl or a benzofused analog thereof, (4)phenyl or naphthyl, and (5) HET¹, wherein HET¹ represents a 5- to10-membered mono- or bicyclic, aromatic or non-aromatic ring, or abenzofused analog thereof, containing 1-3 heteroatoms selected from O, Sand N, groups (2), (3) and (5) above are optionally substituted with 1-2oxo groups, groups (2)-(5) above are further optionally substituted with1-3 substituents independently selected from the group consisting of:(a) halo, (b) nitro, (c) hydroxy, (d) C₁₋₄ alkyl, (e) C₁₋₄alkoxy, (f)C₁₋₄alkylthio, (g) C₃₋₆cycloalkyl, (h) phenyl or naphthyl, (i) phenoxy,(j) benzyl, (k) benzyloxy, and (l) a 5 or 6-membered aromatic ornon-aromatic ring containing from 1-3 heteroatoms selected from O, S andN, groups (d)-(g) above are optionally substituted with oxo and 1-3substituents independently selected from halo and C₁₋₄alkoxy, groups(h)-(l) above are optionally substituted with 1-3 substituentsindependently selected from halo and C₁₋₄alkyl, and group (4) is furtheroptionally substituted up to its maximum with halo groups; R¹ and R² areindependently selected from the group consisting of: (1) H, (2) halo,(3) hydroxy, (4) nitro, (5) cyano, (6) C₁₋₁₀alkyl, C₃₋₁₀cycloalkyl,C₁₋₁₀alkoxy, —S(O)₀₋₂ C₁₋₁₀alkyl or —NHC₁₋₁₀ alkyl, each optionallysubstituted with 1-2 oxo or carboxy groups and further optionallysubstituted with 1-3 substituents independently selected from the groupconsisting of: (a) halo, (b) hydroxy, (c) cyano, (d) C₁₋₄ alkoxy, (e)—NHR⁷, wherein R⁷ is H or C₁₋₅alkyl, said C₁₋₅alkyl optionallysubstituted with —NHR⁸, wherein R⁸ is C₁₋₅alkyl optionally substitutedwith oxo and further optionally substituted with a 5- to 10-memberedmono- or bicyclic, aromatic or non-aromatic ring, or a benzofused analogthereof, containing 1-3 heteroatoms selected from O, S and N, andoptionally substituted with oxo, (f) —S(O)₀₋₂ C₁₋₄alkyl, and (g) HET²,wherein HET² represents a 5- to 7-membered aromatic or non-aromatic ringcontaining 1-4 heteroatoms selected from O, S and NR⁷, wherein R⁷ is Hor C₁₋₅alkyl, said HET² being optionally substituted with oxo andfurther optionally substituted with 1-2 substituents independentlyselected from halo and C₁₋₄alkyl, said C₁₋₄alkyl being optionallysubstituted with 1-3 halo groups, (7) phenoxy or —S(O)₀₋₂-phenyl, (8)benzyloxy or —S(O)₀₋₂-benzyl, (9) benzoyl, (10) phenyl or naphthyl, (11)—O-HET² or —S-HET², said HET² being optionally substituted with oxo andfurther optionally substituted as defined below, and (12) HET³, whereinHET is a 5- or 6-membered aromatic or non-aromatic ring, or a benzofusedanalog thereof, containing from 1 to 4 heteroatoms selected from O, Sand N, said HET³ being optionally substituted with oxo and furtheroptionally substituted as defined below, groups (7)-(12) above are eachoptionally substituted with 1-2 substituents independently selected fromthe group consisting of: halo, cyano, C₁₋₄alkyl and C₁₋₄alkoxy, saidC₁₋₄alkyl and C₁₋₄alkoxy being optionally substituted with 1-3 halogroups; or R¹ and R² may be taken in combination and represent a fusedring as shown below:

wherein Y and X are independently selected from the group consisting of—C(R¹⁰)₂, —C(R¹⁰)₂C(R¹⁰)₂—, —NR¹¹—, —O— and —S—, R³ is as defined below,each R⁹ is independently selected from H and C₁₋₄alkyl, each R¹⁹ isindependently selected from H and C₁₋₄alkyl, and R¹¹ is H or C₁₋₄alkyl,or one R⁹ may be joined with either one R¹⁰ or R¹¹ on an adjacent atomto form a double bond; R³ is C₁₋₁₀alkyl, optionally substituted with 1-2oxo or carboxy groups and further optionally substituted with 1-3substituents independently selected from the group consisting of: (a)halo, (b) hydroxy, (c) cyano, (d) C₁₋₄ alkoxy, (e) —NHR⁷, wherein R⁷ isH or C₁₋₅alkyl, said C₁₋₅alkyl optionally substituted with —NHR⁸,wherein R⁸ is C₁₋₅alkyl optionally substituted with oxo and furtheroptionally substituted with a 5- to 10-membered mono- or bicyclic,aromatic or non-aromatic ring, or a benzofused analog thereof,containing 1-3 heteroatoms selected from O, S and N, and optionallysubstituted with oxo, (f) —S(O)₀₋₂ C₁₋₄alkyl, and (g) HET², wherein HET²represents a 5- to 7-membered aromatic or non-aromatic ring containing1-4 heteroatoms selected from O, S and NR⁷, wherein R⁷ is H orC₁₋₅alkyl, said HET² being optionally substituted with oxo and furtheroptionally substituted with 1-2 substituents independently selected fromhalo or C₁₋₄alkyl, said C₁₋₄alkyl being optionally substituted with 1-3halo groups, each R⁴ is independently selected from the group consistingof: H, halo, hydroxy, C₁₋₆alkyl and C₁₋₄alkoxy, said C₁₋₆alkyl andC₁₋₄alkoxy being optionally substituted with oxo and further optionallysubstituted with 1-3 halo groups; and R⁵ is selected from the groupconsisting of: H, phenyl, naphthyl, C₁₋₆alkyl optionally substitutedwith OR¹² and 1-3 halo groups, and C₅₋₇cycloalkyl optionally containingone heteroatom selected from O, S and NR¹³, wherein R¹² is selected fromthe group consisting of: H, C₁₋₅alkyl optionally substituted with 1-3halo groups, and benzyl optionally substituted with 1-3 substituentsindependently selected from halo, C₁₋₄alkyl and C₁₋₄alkoxy, and R¹³ is Hor C₁₋₄alkyl optionally substituted with 1-3 halo groups; and R⁶represents H; or in the alternative, R⁵ and R⁶ are taken in combinationand represent a ring of 4-7 members, said ring optionally containing oneheteroatom selected from O, S and NR¹³;a compound (aspartic acid analog as caspase-1 (ICE) inhibitor) havingFormula 17.4 (referred to herein as caspase-1 inhibitor having Formula17.4):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein, R₂ is H or alkyl; R₃ is aleaving group such as halogen; R₁ is heteroaryl-CO or an amino acidresidue;a compound (peptidic ketone as ICE inhibitor) having Formula 17.5(referred to herein as caspase-1 inhibitor having Formula 17.5):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein n is 0-2; each AA isindependently L-valine or L-alanine; R₁ is selected from the groupconsisting of N-benzyloxycarbonyl and other groups; R₈, R₉, R₁₀ are eachindependently hydrogen, lower alkyl and other groups;a compound (peptide phenylalkyl ketone as reversible inhibitor of ICE)having Formula 17.6 (referred to herein as caspase-1 inhibitor havingFormula 17.6):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof;a compound (peptide acyloxymethyl ketone) having Formula 17.7 (referredto herein as caspase-1 inhibitor having Formula 17.7):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein Ar is COPh-2,6-(CF₃)₂,COPh-2,6-(CH₃)₂, Ph-F₅ and other groups;a compound (PI aspartate-based peptideα-((2,6-dichlorobenzoyl)oxy)methyl ketone as a potent time-dependentinhibitor of ICE) having Formula 17.8 (referred to herein as caspase-1inhibitor having Formula 17.8):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof;a compound (activated ketone as a potent reversible inhibitor of ICE)having Formula 17.9 (referred to herein as caspase-1 inhibitor havingFormula 17.9):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein X is NH(CH₂)₂, OCO(CH₂)₂,S(CH₂)₃ and other groups;a compound ((1-phenyl-3-(trifluoromethyl)pyrazol-5-yl)oxy)methyl ketoneas an irreversible inhibitor of ICE) having Formula 17.10 (referred toherein as caspase-1 inhibitor having Formula 17.10):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof;a compound (N-acyl-aspartic acid ketone) having Formula 17.11 (referredto herein as caspase-1 inhibitor having Formula 17.11):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein XR₂ is NH(CH₂)₂ Ph,OCO(CH₂)₂ cyclohexyl and other groups;a compound (inhibitor of ICE exemplified by N-acyl-aspartylaryloxymethyl ketone) having Formula 17.12 (referred to herein ascaspase-1 inhibitor having Formula 17.12):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof;a compound (aspartyl α-((diphenylphosphinyl)oxy)methyl ketone as anirreversible inhibitor of ICE) having Formula 17.13 (referred to hereinas caspase-1 inhibitor having Formula 17.13):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof;a compound (α-((tetronoyl)oxy)- and α-((tetramoyl)oxy)methyl ketones asinhibitors of ICE) having Formula 17.14 and 17.15, respectively(referred to herein as caspase-1 inhibitor having Formula 17.14 and17.15, respectively):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof;a compound (peptidomimetic aminomethylene ketone as inhibitor of ICE)having Formula 17.16 (referred to herein as caspase-1 inhibitor havingFormula 17.16):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof;a compound (peptide based ICE inhibitor with the P1 carboxyl groupconverted to an amide) having Formula 17.17 (referred to herein ascaspase-1 inhibitor having Formula 17.17):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof;a compound having Formula 17.18 (referred to herein as caspase-1inhibitor having Formula 17.18):

R-A₁-A₂-X-A₃  Formula 17.18

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein R is a protecting group oroptionally substituted benzyloxy; A₁ is an α-hydroxy or α-amino acidresidue or a radical shown below:

wherein ring A is optionally substituted by hydroxy or C₁₋₄ alkoxy andR_(a) is CO or CS; A₂ is an α-hydroxy or .α-amino acid residue or A₁ andA₂ form together a pseudo-dipeptide or a dipeptide mimetic residue; X isa residue derived from Asp; A₃ is —CH₂—X₁—CO—Y₁, —CH₂—O—Y₂, —CH₂—S—Y₃,wherein X₁ is O or S; Y₁, Y₂ or Y₃ is cycloaliphatic residue, andoptionally substituted aryl;a compound (dipeptide) having Formula 17.19 (referred to herein ascaspase-1 inhibitor having Formula 17.19):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein R₁ is an N-terminalprotecting group; AA is a residue of any natural or non-natural α-aminoacid, β-amino acid, derivatives of an α-amino acid or β-amino acid; R₂is H or CH₂R₄ where R₄ is an electronegative leaving group, and R₃ isalkyl or H, provided that AA is not His, Tyr, Pro or Phe;a compound (dipeptide) having Formula 17.20 (referred to herein ascaspase-1 inhibitor having Formula 17.20):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein R₁ is an N-terminalprotecting group; AA is a residue of a non-natural α-amino acid orβ-amino acid; R₂ is an optionally substituted alkyl or H. Exemplaryinhibitors of caspases and apoptosis include Boc-Phg-Asp-fnk,Boc-(2-F-Phg)Asp-fmk, Boc-(F₃-Val)-Asp-fmk, Boc-(3-F-Val)Asp-fmk,Ac-Phg-Asp-fmk, Ac-(2-F-Phg)-Asp-fmk, Ac-(F₃-Val)-Asp-fmk,Ac-(3-F-Val)-Asp-fmk, Z-Phg-Asp-fmk, Z-(2-F-Phg)-Asp-fmk,Z-(F₃-Val)-Asp-fin, Z-Chg-Asp-fmk, Z-(2-Fug)-Asp-fmk,Z-(4-F-Phg)-Asp-fmk, Z-(4-Cl-Phg)-Asp-fmk, Z-(3-Thg)-Asp-fmk,Z-(2-Fua)-Asp-fmk, Z-(2-Tha)-Asp-fmk, Z-(3-Fua)-Asp-fmk,Z-(3-Tha)-Asp-fmk, Z-(3-Cl-Ala)-Asp-fmk, Z-(3-F-Ala)-Asp-fmk,Z-(3-Ala)Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fmk, Z-(3-C-3-F-Ala)-Asp-fmk,Z-(2-Me-Val)Asp-fmk, Z-(2-Me-Ala)-Asp-fmk, Z-(2-i-Pr-β-Ala)-Asp-fmk,Z-(3-Ph-β-Ala)-Asp-fmk, Z-(3-CN-Ala)-Asp-fmk, Z-(1-Nal)-Asp-fmk,Z-Cha-Asp-fmk, Z-(3-CF₃-Ala)-Asp-fmk, Z-(4-CF₃-Phg)-Asp-fmk,Z-(3-Me₂N-Ala)-Asp-fmk, Z-(2-Abu)-Asp-fmk, Z-Tle-Asp-fmk, Z-Cpg-Asp-fmk,Z-Cbg-Asp-fmk, Z-Thz-Asp-fmk, Z-(3-F-Val)-Asp-fmk, andZ-(2-Thg)-Asp-fmk; where Z is benzyloxycarbonyl, BOC istert-butoxycarbonyl, Ac is acetyl, Phg is phenylglycine, 2-F-Phg is(2-fluorophenyl)glycine, F₃-Val is 4,4,4-trifluorovaline, 3-F-Val is3-fluoro-valine, 2-Thg is (2-thienyl)glycine, Chg is cyclohexylglycine,2-Fug is (2-furyl)glycine, 4-F-Phg is (4-fluorophenyl)glycine, 4-Cl-Phgis (4-chlorophenyl)glycine, 3-Thg is (3-thienyl)glycine, 2-Fua is(2-furyl)alanine, 2-Tha is (2-thienyl)alanine, 3-Fua is(3-furyl)alanine, 3-Tha is (3-thienyl)alanine, 3-Cl-Ala is3-chloroalanine, 3-F-Ala is 3-fluoroalanine, F₃-Ala is3,3,3-trifluoroalanine, 3-F-3-Me-Ala is 3-fluoro-3-methylalanine,3-C₁₋₃-F-Ala is 3-chloro-3-fluoroalanine, 2-Me-Val is 2-methylvaline,2-Me-Ala is 2-methylalanine, 2-i-Pr-β-Ala is3-amino-2-isopropylpropionic acid, 3-Ph-β-Ala is3-amino-3-phenylpropionic acid, 3-CN-Ala is 3-cyanoalanine, 1-Nal is3-(1-naphthyl)-alanine, Cha is cyclohexylalanine, 3-CF₃-Ala is2-amino-4,4,4-trifluorobutyric acid, 4-CF₃-Phg is4-trifluoromethylphenylglycine, 3-Me₂N-Ala is 3-dimethylamino-alanine,2-Abu is 2-aminobutyric acid, Tle is tert-leucine, Cpg iscyclopentylglycine, Cbg is cyclobutylglycine, and Thz is thioproline;compound having Formula 17.21 (referred to herein as caspase-1 inhibitorhaving Formula 17.21):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein R₁ is an N-terminalprotecting group; AA is a residue of any natural or non-natural α-aminoacid, β-amino acid, derivatives of an α-amino acid or β-amino acid; R₂is H or CH₂R₄ where R₄ is an electronegative leaving group; and R₃ isalkyl or H; examples of such caspase-1 inhibitors includeBoc-Ala-Asp-CH₂F, Boc-Val-Asp-CH₂F, Boc-Leu-Asp-CH₂F, Ac-Val-Asp-CH₂F,Ac-Ile-Asp-CH₂F, Ac-Met-Asp-CH₂F, Cbz-Val-Asp-CH₂F, Cbz-#-Ala-Asp-CH₂F,Cbz-Leu-Asp-CH₂F, Cbz-Ile-Asp-CH₂F, Boc-Ala-Asp(OMe)-CH₂F,Boc-Val-Asp(OMe)-CH₂F, Boc-Leu-Asp(OMe)-CH₂F, Ac-Val-Asp(OMe)-CH₂F,Ac-Ile-Asp(OMe)-CH₂F, Ac-Met-Asp(OMe)-CH₂F, Cbz-Val-Asp(OMe)-CH₂F,Cbz-g-Ala-Asp(OMe)-CH₂F, Cbz-Leu-Asp(OMe)CH₂F or Cbz-Ile-Asp(OMe)-CH₂F;compound having Formula 17.22 (referred to herein as caspase-1 inhibitorhaving Formula 17.22):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein R₁ is an N-terminalprotecting group; AA is a residue of a non-natural α-amino acid orβ-amino acid; and R₂ is an optionally substituted alkyl or H; examplesof such caspase-1 inhibitors include Boc-Phg-Asp-fmk,Boc-(2-F-Phg)-Asp-fmk, Boc-(F₃-Val)-Asp-fmk, Boc-(3-F-Val)-Asp-fmk,Ac-Phg-Asp-fmk, Ac-(2-F-Phg)-Asp-fmk, Ac-(F₃-Val)-Asp-fmk,Ac-(3-F-Val)Asp-fmk, Z-Phg-Asp-fmk Z-(2-F-Phg)-Asp-fmk,Z-(F₃-Val)-Asp-fmk, Z-Chg-Asp-fmk, Z-(2-Fug)-Asp-fmk,Z-(4-F-Phg)-Asp-fmk, Z-(4-Cl-Phg)-Asp-fmk, Z-3-Thg)-Asp-fmk,Z-(2-Fua)-Asp-fmk, Z-(2-Tha)-Asp-fmk, Z-3-Fua)-Asp-fmk,Z-(3-Tha)-Asp-fmk, Z-(3-Cl-Ala)-Asp-fmk, Z-(3-F-Ala)-Asp-fmk,Z-(F₃-Ala)-Asp-fmk, Z-(3-F-3-Me-Ala)-Asp-fmk, Z-(3-C₁₋₃-F-Ala)-Asp-fmk,Z-(2-Me-Val)Asp-ink, Z-(2-Me-Ala)-Asp-fmk, Z-(2-i-Pr-β-Ala)-Asp-fmk,Z-(3-Ph-β-Ala)-Asp-fmk, Z-(3-CN-Ala)-Asp-fmk, Z-(1-Nal)-Asp-fmk,Z-Cha-Asp-fmk, Z-3-CF₃-Ala)Asp-fmk, Z-(4-CF₃-Phg)-Asp-fmk,Z-(3-Me₂N-Ala)-Asp-fmk, Z-(2-Abu)-Asp-ink, Z-Tle-Asp-fmk, Z-Cpg-Asp-fmk,Z-Cbg-Asp-fmk, Z-Thz-Asp-fmk, Z-(3-F-Val)-Asp-fmk, and Z-2-Thg)Asp-fmk.

18. Caspase-1 Inhibitor Having Formula 18

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2009/0149436 and US2004/0242494 (incorporated by referencein their entireties). Preferred compounds described in publishedUS2009/0149436 and US2004/0242494 for use in the methods of the presentinvention are referred to herein as caspase-1 inhibitor having Formula18A and 18B, respectively:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein a porphyrin ring structureor a choline ring structure is coordinated to a cobalt atom, L₁ and L₂are each independently, any ligand relative to cobalt, which may or maynot be present, and when it is present, each independently, for example,H₂O, a cyano group, a hydroxyl group, a methyl group, an imidazolylgroup or an adenosyl group;a compound having Formula 18.1 (referred to herein as caspase-1inhibitor having Formula 18.1):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein the compound is a“cobyrinic acid derivative”, each R₁ is independently, for example, ahydroxyl group, an amino group or a lower alkoxy group having 1 to 6carbon atoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy,neopentyloxy, hexyloxy etc.), R₂ is, for example, a hydroxyl group, anamino group, an optionally substituted alkylamino group or an optionallysubstituted or esterified hydroxyalkylamino group, and L₁ and L₂ areeach independently any ligand relative to cobalt, which may or may notbe present, and when it is present, each independently, for example,H₂O, a cyano group, a hydroxyl group, a methyl group, an imidazolylgroup or an adenosyl group—the “alkylamino group” of the above-mentioned“optionally substituted alkylamino group” is an amino group having 1 or2 lower alkyl groups having 1 to 6 carbon atoms (e.g., methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, hexyl and the like) and, for example, methylamino,ethylamino, propylamino, isopropylamino, butylamino, isobutylamino,sec-butylamino, tert-butylamino, pentylamino, isopentylamino,neopentylamino, hexylamino and the like can be mentioned, the“hydroxyalkylamino group” of the above-mentioned “optionally substitutedor esterified hydroxyalkylamino group” means the above-mentioned“alkylamino group” having a hydroxyl group at a substitutable positionand, for example, hydroxymethylamino, 1-hydroxyethylamino,2-hydroxyethylamino, 1-hydroxypropylamino, 2-hydroxypropylamino,3-hydroxypropylamino and the like can be mentioned, the substituent andthe number thereof that the hydroxyalkylamino group may have are notparticularly limited, the hydroxy moiety of the hydroxyalkylamino groupmay further form an ester with α-D-ribofuranose 3-phosphoric acid,imidazolyl-α-D-ribofuranose 3-phosphoric acid,5,6-dimethylbenzimidazolyl-α-D-ribofuranose 3-phosphoric acid and thelike; examples of these cobyrinic acid derivatives include, but notlimited to, cobinamide, cobamide, cobyrinamide, cobyrinic acid, cobyricacid, cobinic acid, cobamic acid and cobalamin, and cobyrinic acidderivatives having a ligand to the cobalt atom of these compounds, suchas dicyanocobinamide, adenosylcobyrinamide, imidazolylcobalamin, cobaltprotoporphyrin, cyanoimidazolylcobamide, cyanocobalamin and the like; a“cobyrinic acid derivative” wherein all R₁ in the above-mentionedformula are amino groups—examples of the cobyric acid derivativesinclude, but not limited to, cobyric acid derivatives such ascobinamide, cobamide, cobyrinamide, cobyric acid, cobalamin and thelike;a compound having Formula 18.2 (referred to herein as caspase-1inhibitor having Formula 18.2):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof; wherein the “cobyric acidderivative” to be used as the caspase-1 inhibitor is dicyanocobinamide.

19. Caspase-1 Inhibitor VX-765 And Related Compounds

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is VX-765 or singlestereoisomers, mixtures of stereoisomers, pharmaceutically acceptablesalts or prodrugs thereof. VX-765 is also known as is(S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylicacid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide. It iscurrently being considered as a treatment for epilepsy.

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2006/0128696 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2006/0128696 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 19:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof

The structure for caspase-1 inhibitor having Formula 19 is meant toinclude all stereochemical forms of the compound; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compound are within the scope of the invention.A preferred isomer is caspase-1 inhibitor having Formula 19A which hasthe “S” configuration at the carbon bearing the tert-butyl group, hasthe “S” configuration at the 2-position of the proline ring, has the “S”configuration at the 3-position of the furanone ring, and has the “R”configuration at the 2-ethoxy position of the furanone ring, as shownbelow.

Another preferred isomer is caspase-1 inhibitor having Formula 19B.

Preparation and purification of VX-765 has been described in U.S. Pat.No. 7,417,029 (herewith incorporated by reference in its entirety).

VX-765 is known as an orally active IL-converting enzyme/caspase-1inhibitor. Recently, Stack et al. described that VX-765 blocked IL-1βsecretion, however, did not disclose its use in a method of the presentinvention (Stack et al., J. Immunol (2005) 175:2630-2634; incorporatedherein by reference)

VX-765 (synthesized by Vertex Pharmaceuticals) can be prepared as a DMSOstock solution and diluted to appropriate concentrations in medium, asdescribed by Stack et al. VX-765 is a prodrug that is converted to itsactive metabolite, VRT-043198, both in vivo and in vitro by enzymaticand hydrolytic cleavage (Stack et al., J. Immunol (2005) 175:2630-2634).VRT-043198 binds to the catalytic site and competitively inhibitsICE/caspase-1 with an inhibition constant (K_(i))=0.8 nM. VX-765inhibits the release of LPS-induced IL-1β and IL-18 by human PBMCs withan IC₅₀ of ˜0.7 μM and reduces inflammatory response in murine models ofinflammatory disease (Stack et al., J. Immunol (2005) 175:2630-2634).

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2006/0128696 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2006/0128696 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 20:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, including

20. Caspase-1 Inhibitor Having Formula 21

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US2006/0128696 (herewith incorporated by reference in itsentirety). Preferred compounds described in published US2006/0128696 foruse in the methods of the present invention are referred to herein ascaspase-1 inhibitor having Formula 21:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, including

21. Caspase-1 Inhibitor Having Formula 22

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished US 2006/0128696 A1 (incorporated by reference in its entirety)and referred to herein as caspase-1 inhibitor having Formula 22:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, including

22. Caspase-1 Inhibitor Having Formula 23 (I), 23 (II) or 23 (III)

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished PCT application WO00/55114 (PCT/US00/06398; herewithincorporated by reference in its entirety). Preferred compoundsdescribed in published PCT application WO00/55114 for use in the methodsof the present invention are referred to herein as caspase-1 inhibitorhaving Formula 23(I), 23(II) or 23(III):

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R₁, is an optionallysubstituted alkyl or hydrogen; R₃ is an N-protecting group; R₂ ishydrogen or optionally substituted alkyl; Q is an optionally substitutedsaturated or partially saturated carbocycle or heterocycle; X is apeptide of 1-4 amino acids or a bond; Y is a peptide of 1-4 amino acidsor a bond; A is CR₆ or nitrogen; B is CR₇ or nitrogen; C is CR₈ ornitrogen; D is CR₉, or nitrogen; provided that not more than two of A,B, C or D is nitrogen; and R₆—R₉, independently are hydrogen, halo,C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl,nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆alkoxy, alkylthio, or carboxy; or one of R₆ and R₇, or R₇ and R₈, or R₈and R₉ are taken together with the carbon atoms to which they areattached to form a carbocycle or heterocycle; E is R₁₄, nitrogen, oxygenor sulfur; F is R₁₅, nitrogen, oxygen or sulfur; G is R₁₆, nitrogen,oxygen or sulfur; provided that only one of E, F, G is nitrogen, oxygenor sulfur and R₁₄—R₁₆ are independently hydrogen, halo, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy;or one of R₁₄ and R₁₅, or R₁₅ and R₁₆, are taken together with thecarbon atoms to which they are attached to form a carbocycle orheterocycle.

23. Caspase-1 Inhibitor Having Formula 24

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished PCT application WO00/61542 (PCT/US00/09319; herewithincorporated by reference in its entirety). Preferred compoundsdescribed in published PCT application WO00/61542 for use in the methodsof the present invention are referred to herein as caspase-1 inhibitorhaving Formula 24:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R₁ is an optionallysubstituted alkyl or hydrogen; R₂ is hydrogen or optionally substitutedalkyl; R₃ is an alkyl, saturated carbocyclic, partially saturatedcarbocyclic, aryl, saturated heterocyclic, partially saturatedheterocyclic or heteroaryl group, wherein said group is optionallysubstituted; X is O, S, NR₄ or (CR₄R₅)_(n) where R₄ and R₅ are, at eachoccurrence, independently selected from the group consisting ofhydrogen, alkyl and cycloalkyl, and n is 0, 1, 2 or 3; or X is NR₄, andR₃ and R₄ are taken together with the nitrogen atom to which they areattached to form a saturated heterocyclic, partially saturatedheterocyclic or heteroaryl group, wherein said group is optionallysubstituted; or X is CR₄R₅, and R₃ and R₄ are taken together with thecarbon atom to which they are attached to form a saturated carbocyclic,partially saturated carbocyclic, aryl, saturated heterocyclic, partiallysaturated heterocyclic or oxygen-containing heteroaryl group, whereinsaid group is optionally substituted; and Y is a residue of a natural ornon-natural amino acid; provided that when X is O, then R₃ is notunsubstituted benzyl or t-butyl; and when X is CH₂, then R₃ is nothydrogen.

24. Caspase-1 Inhibitor Having Formula 25

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 6,632,962 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 6,632,962 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 25:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein X is F or Cl; R¹ is COOH,COO(alkyl), or an isostere thereof; and R² is an aryl group.

25. Caspase-1 Inhibitor Having Formula 26

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished PCT application WO01/16093 (PCT/US00/23566; herewithincorporated by reference in its entirety). Preferred compoundsdescribed in published PCT application WO01/16093 for use in the methodsof the present invention are referred to herein as caspase-1 inhibitorhaving Formula 26:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R₁ is an optionallysubstituted alkyl or hydrogen; R₂ is hydrogen or optionally substitutedalkyl; R₃ and R₄ independently are hydrogen, optionally substitutedaryl, optionally substituted heterocycle, optionally substitutedheteroaryl, optionally substituted carbocyclic, optionally substitutedalkyl, optionally substituted alkenyl, or optionally substitutedalkynyl; R₅ is an optionally substituted alkyl, optionally substitutedcarbocyclic, optionally substituted heterocycle, optionally substitutedaryl or optionally substituted heteroaryl; Z is O, S, NR₈, or(CR₉R₁₀)_(n), where R₈, R₉ and R₁₀ independently are hydrogen, alkyl orcycloalkyl, and n is 0, 1, 2, or 3; and X is a peptide of 1-2 aminoacids or a bond.

26. Caspase-1 Inhibitor Having Formula 27

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 7,517,987 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 7,517,987 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 27:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein where R¹ is hydrogen, CN,CHN₂, R, or —CH₂Y; R is an aliphatic group, a substituted aliphaticgroup, an aryl group, a substituted aryl group, an aralkyl group, asubstituted aralkyl group, a non-aromatic heterocyclic group or asubstituted non-aromatic heterocyclic group; Y is an electronegativeleaving group or —OR, —SR, —OC═O(R), or —OPO (R⁸) (R⁹); R⁸ and R⁹ areindependently selected from R or OR; R² is CO₂H, CH₂CO₂H, or esters,amides or isosteres thereof; R³ is hydrogen or a C₁₋₆ straight chainedor branched alkyl; Ring A contains zero to two double bonds, and isoptionally fused to a saturated or unsaturated five to seven memberedring containing zero to three heteroatoms; X₁ and X₃ in Ring A areindependently selected from nitrogen or carbon, and X₂ is selected froma valence bond, oxygen, sulfur, nitrogen or carbon, wherein any X withsuitable valence may bear a substituent; each carbon with suitablevalence in Ring A, including the fused ring if present, is independentlysubstituted by hydrogen, halo, R, OR, SR, OH, NO₂, CN, NH₂, NHR, N(R)₂,NHCOR, NHCONHR, NHCON(R)₂, NRCOR, NHCO₂R, CO₂R, CO₂H, COR, CONHR,CON(R)₂, S(O)₂R, SONH₂, S(O)R, SO₂NHR, NHS(O)₂R, ═O, ═S, ═NNHR, ═NNR₂,═N—OR, ═NNHCOR, ═NNHCO₂R, ═NNHSO₂R, or ═NR; each substitutable nitrogenin Ring A is substituted by hydrogen, R, COR, S(O)₂R, or CO₂R; providedthat when X₃ is a carbon, a substituent on X₃ is attached by an atomother than nitrogen, and further provided that at least one X in Ring Ais a nitrogen.

27. Caspase-1 Inhibitor Having Formula 28

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. Nos. 6,689,784 and 7,074,782 (herewith incorporated by reference intheir entireties).). Preferred compounds described in U.S. Pat. Nos.6,689,784 and 7,074,782 for use in the methods of the present inventionare referred to herein as caspase-1 inhibitor having Formula 28:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein Z is oxygen or sulfur; R¹is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y; R is a C₁₋₁₂aliphatic, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl; Y is anelectronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is a group capable of fitting into the S2 sub-siteof a caspase; R⁴ and R⁵ taken together with the intervening nitrogenform a mono-, bi- or tricyclic hetero ring system having 1-6 heteroatomsselected from nitrogen, oxygen or sulfur.

28. Caspase-1 Inhibitor Having Formula 29

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 7,053,057 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 7,053,057 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 29:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein Ring A is an optionallysubstituted piperidine, tetrahydroquinoline or tetrahydroisoquinolinering; R¹ is hydrogen, CHN₂, R, or —CH₂Y; R is an optionally substitutedgroup selected from an aliphatic group, an aryl group, an aralkyl group,a heterocyclic group, or an heterocyclylalkyl group; Y is anelectronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; Ar is an optionally substituted aryl group; and R³ ishydrogen, an optionally substituted C₁₋₆ alkyl, F₂, CN, aryl or R³ isattached to Ar to form an unsaturated or partially saturated five or sixmembered fused ring having 0-2 heteroatoms.

29. Caspase-1 Inhibitor Having Formula 30

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 7,205,327 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 7,205,327 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 30:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is COH, CH₂CO₂H, oresters, amides or isosteres thereof; R² is hydrogen or an optionallysubstituted C₁-C₆ aliphatic group; R³ is hydrogen or an optionallysubstituted C₁-C₆ aliphatic group; and R⁴ and R⁵ are each independentlyselected from hydrogen, an optionally substituted C₁˜C₆ aliphatic group,or R⁴ and R⁵ taken together with the ring to which they are attachedform an optionally substituted bicyclic ring, said bicyclic ringselected from the following:

30. Caspase-1 Inhibitor Having Formula 31A Or 31B

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 6,184,210 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 6,184,210 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 31A:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R₁ is an N-terminalprotecting group; AA is a residue of any natural or non-natural α-aminoacid, β-amino acid, derivatives of an α-amino acid or β-amino acid; R₂is H or CH₂ R₄ where R₄ is an electronegative leaving group, and R₃ isalkyl or H, provided that AA is not His, Tyr, Pro or Phe.

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 6,184,210 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 6,184,210 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 31B:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R₁ is an N-terminalprotecting group selected from the group consisting of t-butoxycarbonyl(Boc), acetyl (Ac) and benzyloxycarbonyl (Cbz); R₃ is alkyl or hydrogen;and AA is a residue of an amino acid selected from the group consistingof valine (Val), isoleucine (Ile) and leucine (Leu).

31. Caspase-1 Inhibitor Having Formula 32

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 6,184,244 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 6,184,244 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 32:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein n is 1 or 2; R¹ is alkyl,cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl,(substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(m) CO₂R⁴, wherein m=1-4, and R⁴ is as defined below; R² is a hydrogen atom,chloro, alkyl, cycloalkyl, cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl,(heteroaryl)alkyl or (CH₂)_(p) CO₂R⁵, wherein p=0-4, and R⁵ is asdefined below; R³ is a hydrogen atom, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁴ is ahydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or(substituted)phenylalkyl; R⁵ is a hydrogen atom, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is anatural or unnatural amino acid; B is a hydrogen atom, a deuterium atom,alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl,phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl,halomethyl, CH₂ ZR⁶, CH₂OCO(aryl), or CH₂OCO(heteroaryl), orCH₂OPO(R⁷)R⁸, where Z is an oxygen, OC(═O) or a sulfur atom; R⁶ isphenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl,heteroaryl or (heteroaryl)alkyl; R⁷ and R⁸ are independently selectedfrom a group consistent of alkyl, cycloalkyl, phenyl, substitutedphenyl, phenylalkyl, (substituted phenyl)alkyl and (cycloalkyl)alkyl;and X and Y are independently selected from the group consisting of ahydrogen atom, halo, trihalomethyl, amino, protected amino, an aminosalt, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl.

32. Caspase-1 Inhibitor Having Formula 33

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 6,187,771 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 6,187,771 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 33:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein n is 1 or 2; m is 1 or 2;A is R² CO—, R3-O—CO—, or R⁴ SO2-; a group of the formula:

further wherein: R¹ is a hydrogen atom, alkyl or phenylalkyl; R² isalkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substitutedphenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl or (substitutedphenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl,phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl,or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl,phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl,heteroaryl, or (heteroaryl)alkyl; R⁶ is alkyl, cycloalkyl,(cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ isalkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substitutedphenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸is an amino acid side chain chosen from the group consisting of naturaland unnatural amino acids; B is a hydrogen atom, a deuterium atom,alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substitutedphenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or ahalomethyl group; a group of the formula: wherein R⁹ is phenyl,substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl,or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom;a group of the formula: —CH₂—)—CO-(aryl);a group of the formula: —CH₂—O—CO-(heteroaryl);a group of the formula: —CH₂—O—PO—(R¹⁰)R¹¹;wherein R¹⁰ and R¹¹ are independently selected from a group consistingof alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl.

33. Caspase-1 Inhibitor Having Formula 34

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 6,197,750 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 6,197,750 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 34:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein A is a natural orunnatural amino acid of Formula IIa-i:

B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, phenyl,substituted phenyl, naphthyl, substituted naphthyl, 2-benzoxazolyl,substituted 2-oxazolyl, (CH₂)cycloalkyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl), (CH₂)_(n)(1 or 2-naphthyl),(CH₂)_(n)(heteroaryl), halomethyl, CO₂R¹², CONR¹³R¹⁴, CH₂ZR¹⁵,CH₂OCO(aryl), CH₂OCO(heteroaryl), or CH₂OPO(R¹⁶)R¹⁷, where Z is anoxygen or a sulfur atom, or B is a group of the Formula IIIa-c:

R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, substituted phenyl,phenylalkyl, substituted phenylalkyl, naphthyl, substituted naphthyl, (1or 2 naphthyl)alkyl, heteroaryl, (heteroaryl)alkyl, R^(1a) (R^(1b))N, orR^(1c) O; and R² is hydrogen, lower alkyl, cycloalkyl,(cycloalkyl)alkyl, phenylalkyl, or substituted phenylalkyl; and wherein:R^(1a) and R^(1b) are independently hydrogen, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substitutedphenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl,heteroaryl, or (heteroaryl)alkyl, with the proviso that R^(1a) andR^(1b) cannot both be hydrogen; R^(1c) is alkyl, cycloalkyl,(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, substitutedphenylalkyl, naphthyl, substituted naphthyl, (1 or 2 naphthyl)alkyl,heteroaryl, or (heteroaryl)alkyl; R³ is C₁₋₆ lower alkyl, cycloalkyl,phenyl, substituted phenyl, (CH₂)_(n)NH₂, (CH₂)_(n)NHCOR⁹,(CH₂)_(n)N(C═NH)NH₂, (CH₂)_(m)CO₂R², (CH₂)_(m)OR10, (CH₂)_(m)SR¹¹,(CH₂)_(n)cyclo alkyl, (CH₂)_(n)phenyl, (CH₂)(substituted phenyl),(CH₂)_(n)(1 or 2-naphthyl) or (CH₂)_(n)(heteroaryl), wherein heteroarylincludes pyridyl, thienyl, furyl, thiazolyl, imidazolyl, pyrazolyl,isoxazolyl, pyrazinyl, pyrimidyl, triazinyl, tetrazolyl, and indolyl;R^(3a) is hydrogen or methyl, or R³ and R^(3a) taken together are—(CH₂)_(d)— where d is an integer from 2 to 6; R⁴ is phenyl, substitutedphenyl, (CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl), cycloalkyl, orbenzofused cycloalkyl; R⁵ is hydrogen, lower alkyl, cycloalkyl, phenyl,substituted phenyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl); R⁶ ishydrogen, fluorine, oxo, lower alkyl, cycloalkyl, phenyl, substitutedphenyl, naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl), (CH₂)_(n)(1 or 2-naphthyl), OR¹⁰, SR¹¹ orNHCOR⁹; R⁷ is hydrogen, oxo (i.e., ═O), lower alkyl, cycloalkyl, phenyl,substituted phenyl, naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl); R⁸ islower alkyl, cycloalkyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl), (CH₂)_(n)(1 or 2-naphthyl), or COR⁹; R⁹is hydrogen, lower alkyl, cycloalkyl, phenyl, substituted phenyl,naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substitutedphenyl), (CH₂)_(n)(1 or 2-naphthyl), OR¹², or NR¹³ R¹⁴; R¹⁰ is hydrogen,lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl,(CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substituted phenyl), or(CH₂)_(n)(1 or 2-naphthyl); R¹¹ is lower alkyl, cycloalkyl, phenyl,substituted phenyl, naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl); R¹² islower alkyl, cycloalkyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl); R¹³ ishydrogen, lower alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl,substituted naphthyl, (CH₂)_(n)cycloalkyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl), or (CH₂)_(n)(1 or 2-naphthyl); R¹⁴ ishydrogen or lower alkyl; or R¹³ and R¹⁴ taken together form a five toseven membered carbocyclic or heterocyclic ring, such as morpholine, orN-substituted piperazine; R¹⁵ is phenyl, substituted phenyl, naphthyl,substituted naphthyl, heteroaryl, (CH₂)_(n)phenyl, (CH₂)_(n)(substitutedphenyl), (CH₂)_(n)(1 or 2-naphthyl), or (CH₂)_(n)(heteroaryl); R¹⁶ andR¹⁷ are independently lower alkyl, cycloalkyl, phenyl, substitutedphenyl, naphthyl, phenylalkyl, substituted phenylalkyl, or(cycloalkyl)alkyl; R¹⁸ and R¹⁹ are independently hydrogen, alkyl,phenyl, substituted phenyl, (CH₂)_(n)phenyl, (CH₂)_(n)(substitutedphenyl), or R¹⁸ and R¹⁹ taken together are —(CH═CH)₂—; R²⁰ is hydrogen,alkyl, phenyl, substituted phenyl, (CH₂)_(n)phenyl,(CH₂)_(n)(substituted phenyl); R²¹, R²² and R²³ are independentlyhydrogen, or alkyl; X is CH₂, (CH₂)₂, (CH₂)₃, or S; Y¹ is O or NR²³; Y²is CH₂, O, or NR²³; a is 0 or 1 and b is 1 or 2, provided that when a is1 then b is 1; c is 1 or 2, provided that when c is 1 then a is 0 and bis 1; m is 1 or 2; and n is 1, 2, 3 or 4.

34. Caspase-1 Inhibitor Having Formula 35

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described in U.S.Pat. No. 6,242,422 (herewith incorporated by reference in its entirety).Preferred compounds described in U.S. Pat. No. 6,242,422 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 35:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein n is 0, 1 or 2; X is CH₂,C═O, O, S or NH; A is a natural or unnatural amino acid of FormulaIIa-i:

B is a hydrogen atom, a deuterium atom, C₁₋₁₀ straight chain or branchedalkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, substitutednaphthyl, 2-benzoxazolyl, substituted 2-oxazolyl, (CH₂)_(m)cycloalkyl,(CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl), (CH₂)_(m)(1 or2-naphthyl), (CH₂)_(m)heteroaryl, halomethyl, CO₂R¹³, CONR¹⁴R¹⁵,CH₂ZR¹⁶, CH₂OCO(aryl), CH₂OCO(heteroaryl), or CH₂OPO(R¹⁷)R¹⁸, where Z isan oxygen or a sulfur atom, or B is a group of the Formula IIIa-c:

R¹ is phenyl, substituted phenyl, naphthyl, substituted naphthyl,heteroaryl, or substituted heteroaryl; R² is hydrogen, alkyl,cycloalkyl, phenyl, substituted phenyl, (CH₂)_(m)NH₂, (CH₂)_(m) NHCOR¹⁰,(CH₂)_(m) N(C═NH)NH₂, (CH₂)_(p)CO₂R³, (CH₂)_(p)OR¹¹, (CH₂)_(p)SR¹²,(CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl),(CH₂)_(m)(1 or 2-naphthyl), or (CH₂)_(m)heteroaryl, wherein heteroarylincludes (but is not limited to) pyridyl, thienyl, furyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, pyrazinyl, pyrimidyl, triazinyl,tetrazolyl, and indolyl; R³ is hydrogen, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenylalkyl, or substituted phenylalkyl; and whereinR⁴ is alkyl, cycloalkyl, phenyl, substituted phenyl, (CH₂)_(m)NH₂,(CH₂), NHCOR¹⁰, (CH₂)_(m) N(C═NH)NH₂, (CH₂)_(p) CO₂R³, (CH₂)_(p)OR¹¹,(CH₂)_(p)SR¹², (CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl,(CH₂)_(m)(substituted phenyl), (CH₂)_(m)(1 or 2-naphthyl), or(CH₂)_(m)heteroaryl, wherein heteroaryl includes (but is not limited to)pyridyl, thienyl, furyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,pyrazinyl, pyrimidyl, triazinyl, tetrazolyl, and indolyl; R^(4a) ishydrogen or methyl, or R⁴ and R^(4a) taken together are —(CH₂)_(d)—where d is an integer from 2 to 6; R⁵ is phenyl, substituted phenyl,(CH₂)_(p)phenyl, (CH₂)_(p)(substituted phenyl), cycloalkyl, orbenzofused cycloalkyl; R⁶ is hydrogen, alkyl, cycloalkyl, phenyl,substituted phenyl, (CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl,(CH₂)_(m)(substituted phenyl), or (CH₂)_(m)(1 or 2-naphthyl); R⁷ ishydrogen, fluorine, oxo (i.e., ═O), alkyl, cycloalkyl, phenyl,substituted phenyl, naphthyl, (CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl,(CH₂)_(m)(substituted phenyl), (CH₂)_(m)(1 or 2-naphthyl), OR¹¹, SR¹²,or NHCOR¹⁰; R⁸ is hydrogen, oxo, alkyl, cycloalkyl, phenyl, substitutedphenyl, naphthyl, (CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl,(CH₂)_(m)(substituted phenyl), or (CH₂)_(m)(1 or 2-naphthyl); R⁹ isalkyl, cycloalkyl, (CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl,(CH₂)_(m)(substituted phenyl), (CH₂)_(m)(1 or 2-naphthyl), or COR¹⁰; R¹⁰is hydrogen, alkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl,(CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl),(CH₂)_(m)(1 or 2-naphthyl), OR¹³, or NR¹⁴ R¹⁵; R¹¹ is hydrogen, alkyl,cycloalkyl, phenyl, substituted phenyl, naphthyl, (CH₂)_(m)cycloalkyl,(CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl), or (CH₂)_(m)(1 or2-naphthyl); R¹² is alkyl, cycloalkyl, phenyl, substituted phenyl,naphthyl, (CH₂)_(m)cycloalkyl, (CH₂), phenyl, (CH₂)_(m)(substitutedphenyl), or (CH₂)_(m)(1 or 2-naphthyl); R¹³ is alkyl, cycloalkyl,(CH₂)_(m)cycloalkyl, (CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl), or(CH₂)_(m)(1 or 2-naphthyl); R¹⁴ is hydrogen, alkyl, cycloalkyl, phenyl,substituted phenyl, naphthyl, substituted naphthyl, (CH₂)_(m)cycloalkyl,(CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl), or (CH₂)41 or2-naphthyl); R¹⁵ is hydrogen or alkyl; or R¹⁴ and R¹⁵ taken togetherform a five, six or seven membered carbocyclic or heterocyclic ring,such as morpholine or N-substituted piperazine; R¹⁶ is phenyl,substituted phenyl, naphthyl, substituted naphthyl, heteroaryl,(CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl), (CH₂)_(m)(1 or2-naphthyl), or (CH₂)_(m)heteroaryl; R¹⁷ and R¹⁸ are independentlyalkyl, cycloalkyl, phenyl, substituted phenyl, naphthyl, or phenylalkyl,substituted phenylalkyl, or (cycloalkyl)alkyl; R¹⁹ and R²⁰ areindependently hydrogen, alkyl, phenyl, substituted phenyl, (CH₂) phenyl,or (CH₂)_(m)(substituted phenyl), or R¹⁹ and R²⁰ taken together are—(CH═CH)²—; R²¹ is hydrogen, alkyl, phenyl, substituted phenyl,(CH₂)_(m)phenyl, (CH₂)_(m)(substituted phenyl); R²², R²³ and R²⁴ areindependently hydrogen or alkyl; Y¹ is CH₂, (CH₂)₂, (CH₂)₃, or S; Y² isO or NR²⁴; Y³ is CH₂, O, or NR²⁴; a is 0 or 1 and b is 1 or 2, providedthat when a is 1 then b is 1; c is 1 or 2, provided that when c is 1then a is 0 and b is 1; m is 1, 2, 3 or 4; and p is 1 or 2.

35. Caspase-1 Inhibitor Having Formula 36

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished PCT application WO2002/22611 (herewith incorporated byreference in its entirety).). Preferred compounds described in publishedPCT application WO2002/22611 for use in the methods of the presentinvention are referred to herein as caspase-1 inhibitor having Formula36:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein Ring A is an optionallysubstituted piperidine, tetrahydroquinoline or tetrahydroisoquinolinering; R¹ is hydrogen, CN, CHN₂, R, or CH₂Y; R is an optionallysubstituted group selected from an aliphatic group, an aryl group, or anaralkyl group; Y is an electronegative leaving group; R² is CO₂H,CH₂CO₂H, or esters, amides or isosteres thereof; and R³ is hydrogen, anoptionally substituted aryl group, an optionally substituted aralkylgroup or an optionally substituted C₁₋₆ aliphatic group, R⁴ is anoptionally substituted group selected from an aryl group or aheterocyclyl group, or R³ and R⁴ taken together with the nitrogen towhich they, are attached optionally form a substituted or unsubstitutedmonocyclic, bicyclic or tricyclic ring.

36. Caspase-1 Inhibitor Having Formula 37

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a compound described inpublished PCT application WO02/085899 (PCT/US02/12638; herewithincorporated by reference in its entirety). Preferred compoundsdescribed in published PCT application WO02/085899 for use in themethods of the present invention are referred to herein as caspase-1inhibitor having Formula 37:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is hydrogen, CN, CHN₂,R, or —CH₂Y; R is an aliphatic group, a substituted aliphatic group, anaryl group, a substituted aryl group, an aralkyl group, a substitutedaralkyl group, a non-aromatic heterocyclic group, or a substitutednon-aromatic heterocyclic group; Y is an electronegative leaving group,—OR, —SR, —OC═O(R), or —OPO (R³) (R⁴); R³ and R⁴ are independently R orOR; R² is CO₂H, CH₂CO₂H, or optionally substituted esters, amides orisosteres thereof; A is C═O or SO₂; X¹ is oxygen, sulfur, —NH, or —CH₂,wherein —NH is optionally substituted by an alkyl group, a cycloalkylgroup, a (cycloalkyl) alkyl group, an amino acid N-terminal protectinggroup, or COR and —CH₂ is optionally substituted by fluorine, an alkylgroup, a cycloalkyl group, a (cycloalkyl) alkyl group, an aralkyl group,an aryl group, an alkyloxy group, an alkylthioxy group, an aryloxygroup, an arylthioxy group, an oxo group (i.e., ═O), or a NHCOR group;X² is oxygen, sulfur, —NH, or —CH₂, wherein —NH is optionallysubstituted by an alkyl group, or an amino acid N-terminal protectinggroup and —CH₂ is optionally substituted by an alkyl group, an arylgroup, an alkyloxy group, an alkylthioxy group, an aryloxy group, anarylthioxy group, or an oxo (i.e., ═O) group, a NHCOR group; X¹ and X²optionally form part of a phenyl ring that is fused to the adjoiningring Q; X³ is CH₂ or X² and X³ optionally form part of a phenyl ringthat is fused to the adjoining ring Q, provided that when X² forms aring with X³, then X² does not form a ring with X¹; any two hydrogensattached to adjacent positions in ring Q are optionally replaced by adouble bond; and Z is an optionally substituted ring selected from thegroup consisting of a carbocyclic, an aryl, a saturated heterocycle, apartially saturated heterocycle, and a heteroaryl wherein the ring isconnected to A at a ring carbon.

37. Caspase-1 Inhibitor From Extremophilic Fungus

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is a caspase-1 inhibitor froman extremophilic fungus or single stereoisomers, mixtures ofstereoisomers, pharmaceutically acceptable salts or prodrugs thereof.Recently, Stierle et al. described caspase-1 inhibitors from anextremophilic fungus that specifically target leukemia cell lines,however, did not disclose its use in a method of the present invention(Stierle et al., J Nat Prod (2012) 75:344-350; Stierle et al., J NatProd (2011) 74(10):2273-2277; Stierle et al., J Nat Prod (2012)75:262-266; incorporated herein by reference in their entireties).

Suitable caspase-1 inhibitors from an extremophilic fungus useful topractice a method of the present invention include those described inFIGS. 30A and 30B or single stereoisomers, mixtures of stereoisomers,pharmaceutically acceptable salts or prodrugs thereof.

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is Berkedrimane B or singlestereoisomers, mixtures of stereoisomers, pharmaceutically acceptablesalts or prodrugs thereof having Formula 38:

Compound of Formula 38 is known as5aS,9S,9aR,9bS)-9b-Hydroxy-6,6,9a-trimethyl-3-oxo-1,3,5,5a,6,7,8,9,9a,9b-decahydronaphtho[1,2-c]furan-9-ylN-acetyl-L-valinate. (ChemSpider ID 26333271).

38. Caspase-1 Inhibitor VRT-018858

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is VRT-018858 or singlestereoisomers, mixtures of stereoisomers, pharmaceutically acceptablesalts or prodrugs thereof. Recently, Ross et al. described VRT-018858 asa selective, non-peptide caspase-1 inhibitor that markedly reduced braindamage induced by transient ischemia in rat, however, did not discloseits use in a method of the present invention (Ross et al.,Neuropharmacology (2007) 53:638-642; incorporated herein by reference inits entirety).

VRT-018858 is the active metabolite of the selective caspase-1 inhibitorpro-drug, pralnacasan (Ross et al., Neuropharmacology (2007)53:638-642). VRT-018858 may be provided, e.g., by VertexPharmaceuticals, Inc. (Cambridge, USA). VRT-018858 is potently selectivefor group I or inflammatory caspases, with K_(i) values againstcaspase-1 and caspase-4 of 1.3 nM and 0.4 nM, respectively (Ross etal.). Further, VRT-018858 exhibits >100-fold selectivity for caspase 1and caspase-4 against other caspases (Ross et al.). Ross et al. describeinjection icy of rats with 2.5, 5, 10, or 20 μg VRT-018858. One of skillin the art will be able to extrapolate such treatment to the treatmentof a patient in methods described herein.

39. Caspase-1 Inhibitor IDN-6556

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is IDN-6556 or singlestereoisomers, mixtures of stereoisomers, pharmaceutically acceptablesalts or prodrugs thereof. IDN-6556(3-{2-(2-tert-Butyl-phenylaminooxalyl)-amino]-propionylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid) is a novel irreversible broad-spectrum caspase inhibitor(with activity against all tested human caspases). It shows noinhibition of other classes of proteases or other enzymes or receptors(Hoglen et al., Pharmacol Exp Ther (2004) 309:634-640).

Recently, Baskin-Bey et al. described phase II clinical trials of usingIDN-6556 in human liver preservation injury however, did not did notdisclose its use in a method of the present invention (Baskin-Bey etal., Am J Transplantation (2007) 7:218-225; incorporated herein byreference in its entirety). Baskin-Bey et al. showed that IDN-6556reduced CI/WR (cold ischemia/warm reperfusion)-induced apoptosis andinjury in the liver and other organs (Baskin-Bey et al., Am JTransplantation (2007) 7:218-225). A phase I clinical trial of IDN-6556administered to patients with hepatic impairment showed the drug to bewell tolerated. In the phase II trial, some patients received IDN-6556(supplied in the University of Wisconsin (UW) orhistidine-tryptophan-ketoglutamate (HTK) solution) at a concentration of15 μg/mL and perfused through the portal vein. Others received IDN-6556in the cold storage and flush solutions at a concentration of 5 μg/mLand following liver transplantation, intravenously (0.5 mg/kg) every 6 hfor 24 h. Yet others received IDN-6556 (15 μg/mL) in the storage andflush solutions administered at a concentration of 0.5 mg/kg every 6 hfor 48 h.

In addition, Pockros et al. (Pockros et al., Hepatology (2007)46(2):324-329; incorporated by reference in its entirety) reported thatorally taken or intravenously administered IDN-6556 was well toleratedand efficacious in clinical trial evaluating aminotransferase activityin patients with chronic Hepatitis C. In this trial, IDN-6556 dosesranged from 5 mg to 400 mg daily, given from 1 to 3 times per day.

Thus, one of ordinary skill in the art, will also appreciate thatIDN-6556 will also be well tolerated in patients being treated accordingto a method of the present invention, particular in patients having anHIV-1 infection or suspected of having an HIV-1 infection or havingAIDS.

40. CRID as Caspase-1 Inhibitors

In a screen for inhibitors of IL-1β production a novel class ofsulfonylurea containing compounds were identified. These so-calledcytokine release inhibitory drugs or CRIDs (CP-424,174 and CP-412,245)inhibited the post-translational processing and secretion of IL-1β inresponse to LPS (lipopolysaccharide) and ATP in human monocytes(Perregaux et al., J Pharmacol Exp Therapeutics (2001) 299:187-197).Further studies identified glutathione-S-transferase omega 1 (GSTO1) asa possible target for CRIDs (Laliberte et al., J Biol Chem (2003)278:16567-16578). More recently Coll and O'Neill characterized theinhibitory activity of the CRID CP-456,773 (termed CRID3) againstmultiple inflammasomes and found that CRID3 inhibited both NLRP3(Nod-like receptor protein) and AIM2 (Absent in melanoma-2)inflammasomes by preventing ASC (adaptor molecule apoptosis-associatedspeck-like protein containing a CARD [caspase activation and recruitmentdomain]) oligomerisation (Coll and O'Neill, PLos ONE 6(12) e29539). Inaddition GSTO1 was found to associate with ASC suggesting that it mightplay a role in inflammasome signaling and could indeed be a target ofCRID3.

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is CP-412-245 having Formula39:

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is CP-424,174 having Formula40:

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is CRID1 having Formula 41:

CRID1 is known as1-(4-Chloro-2,6-diisopropylphenyl)-3-[2-fluoro-5-oxiranylbenzenesulfonyl]urea (CP-452,759; Laliberte et al., J Biol Chem (2003)278(19):16567-16578).

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is CRID2 having Formula 42:

CRID2 is known as1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[2-fluoro-5-oxiranylbenzenesulfonyl]urea(CP-470,947; Laliberte et al., J Biol Chem (2003) 278(19):16567-16578).

In some embodiments of the present invention a caspase-1 inhibitor foruse in a method of the present invention is CRID3. CP-456,773 (CRID3)can be obtained from Amgen, Inc. (Thousand Oaks, Calif., USA) and hasFormula 43.

Preparation and purification of CP-456,773 (CRID 3) has been described(Laliberte et al., J Biol Chem (2003) 278(19):16567-16578).

CP-412,245, CP-424,174, CRID1, CRID2, and CRID3 can be obtained, e.g.,from Pfizer, Inc. (Groton, Conn., USA).

B. Testing Inhibitors

The present invention describes a variety of caspase-1 inhibitors foruse in the methods of the present invention. These inhibitors are usefulas pharmaceutical agents, especially in the treatment of HIV-1 infectionand AIDS and in methods inhibiting death of CD4 T-cells as more fullydescribed herein. Pharmaceutically acceptable salts of the compoundsdisclosed herein can be used to practice the present invention.

The caspase-1 inhibitors described herein and agents derived therefromthrough routine chemical manipulations that are useful for practicingthe present invention can be tested for their potential to inhibit theactivation and/or activity of caspase-1 using assays described herein(see Examples).

Other useful caspase-1 activity assays and caspase-1 inhibitor assaysare available from R&D Systems (Minneapolis, Minn., USA) and are alsodescribed in Winter et al. (Winter et al., Anticancer Res (2004)24:1377-1386).

IV. Methods

The caspase-1 inhibitors described herein and agents derived therefromthrough routine chemical manipulations are useful for practicing themethods described below. They will be set forth exemplary once forpracticing the method for the treatment of HIV-1 infection and/or AIDS.One of skill in the art will appreciate that the caspase-1 inhibitorsdescribed herein, can also be used for practicing the other methodsdescribed herein.

A. Treatment of HIV-1 Infection and/or AIDS

The present invention provides methods for the treatment of an HIV-1infection and/or AIDS. The present invention also provides a method forthe treatment of a patient having an HIV-1 infection or suspected ofhaving an HIV-1 infection or having AIDS. These methods comprise thestep of selecting a patient having an HIV-1 infection or suspected ofhaving an HIV-1 infection or having AIDS and administering to thepatient a compound of the invention. Thereby the patient is treated.

In some embodiments of the present invention, the method for thetreatment of a patient having an HIV-1 infection or suspected of havingan HIV-1 infection or having AIDS comprises the step of administering tothe patient having an HIV-1 infection or suspected of having an HIV-1infection or having AIDS a caspase-1 inhibitor. Thereby the patient istreated.

The invention allows the selection of patients for treatment with acompound described herein, based on an appreciated need of the patientfor a treatment of HIV-1 infection or for a treatment of AIDS. In someembodiments this method comprises the step of selecting a patient havingan HIV-1 infection or suspected of having an HIV-1 infection or havingAIDS and administering to the patient a caspase-1 inhibitor. Thereby thepatient is treated.

In some embodiments, the method comprises the step of selecting apatient on the basis of that patient being in need of the inhibition ofcaspase-1 for the treatment of the HIV-1 infection or AIDS.

In some embodiments of methods for the treatment of HIV-1 infectionand/or AIDS, the method comprises administering to a patient in need ofsuch treatment, an effective amount of a caspase-1 inhibitor or apharmaceutically acceptable salt, prodrug or active derivative of such asubstance. The substance in question has been identified as one that iscapable of treating HIV-1 infection or AIDS in a patient by their effecton inhibition of activity of caspase-1.

In some embodiments of the method for treating an HIV-1 infection and/orAIDS in a patient, the caspase-1 inhibitor is a caspase-1 inhibitorhaving Formula 1a or 1b. In some embodiments, the caspase-1 inhibitor isa caspase-1 inhibitor having Formula 2. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 3. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 4, 4.1, 4.2, or 4.3. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 5. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 6. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 7. In some embodiments, the caspase-1 inhibitoris a caspase-1 inhibitor having Formula 8, 8.1, or 8.2. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 9 or 9.1. In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 10. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 11. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 12. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 13. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 14. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 15. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, or16.7. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7,17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15, 17.16, 17.17,17.18, 17.19, 17.20, 17.21, or 17.22. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 18A, 18B, 18.1, or18.2. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 19, 19A, or 19B. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 20 or 20A.In some embodiments, the caspase-1 inhibitor is a caspase-1 inhibitorhaving Formula 21 or 21A. In some embodiments, the caspase-1 inhibitoris a caspase-1 inhibitor having Formula 22 or 22A. In some embodiments,the caspase-1 inhibitor is a caspase-1 inhibitor having Formula 23(I),23(II), or 23(III). In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 24. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 25. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 26. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 27. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 28. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 29. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 30. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 31A or 31B. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 32. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 33. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 34. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 35. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 36. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 37. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 38. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 39. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 40. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 41. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 42. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 43. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor selectedfrom the compounds depicted in FIGS. 30 and 31. In some embodiments, thecaspase-1 inhibitor is a compound disclosed in any of the published EPpatent applications, in any of published PCT patent applications, in anyof published U.S. patent applications, or in any of the granted U.S.patents disclosed herein and incorporated herein by reference in theirentireties.

In some embodiments of the method for treating an HIV-1 infection and/orAIDS in a patient, the caspase-1 inhibitor is selected from the group ofcaspase-1 inhibitors having Formula 1a, 1b, 2, 3, 4, 4.1, 4.2, 4.3, 5,6, 7, 8, 8.1, 8.2, 9, 9.1, 10, 11, 12, 13, 14, 15, 16, 16.1, 16.2, 16.3,16.4, 16.5, 16.6, 16.7, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7,17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15, 17.16, 17.17,17.18, 17.19, 17.20, 17.21, 17.22, 18A, 18B, 18.1, 18.2, 19, 19A, 19B,20, 20A, 21, 21A, 22, 22A, 23(I), 23(II), 23(III), 24, 25, 26, 27, 28,29, 30, 31A, 31B, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, acaspase-1 inhibitor depicted in FIGS. 30 and 31, and combinationsthereof.

In some embodiments of this method, the method the patient comprisescells having incomplete HIV-1 nucleic acids.

Formulation, administration, therapeutic effective amounts and dosing ofpharmaceutical compositions useful in methods for the treatment of HIV-1infection and AIDS using a compound of the present invention aredescribed below.

In some embodiments of the method for treating an HIV-1 infection and/orAIDS, the method comprises the step of selecting a patient who hasdeveloped a resistance against an antiviral HIV-1 drug. Resistance of anHIV-1 infected patient to an antiviral HIV-1 drug typically isdetermined by a treating clinician.

In some embodiments of this method, the method comprises the step ofadministering to the patient an anti HIV-1 compound, such as a HAARTcompound and as described further below.

In some embodiments of the method for treating an HIV-1 infection and/orAIDS, the method comprises the step of selecting a patient who has areduced total lymphocyte count of less than 1,000/mm³. In someembodiments of the method for treating an HIV-1 infection and/or AIDS,the method comprises the step of selecting a patient who has a reducedtotal lymphocyte count of less than 750/mm³. In some embodiments of themethod for treating an HIV-1 infection and/or AIDS, the method comprisesthe step of selecting a patient who has a reduced total lymphocyte countof less than 500/mm³. In some embodiments of the method for treating anHIV-1 infection and/or AIDS, the method comprises the step of selectinga patient who has a reduced T-cell count of less than 500/mm³. In someembodiments of the method for treating an HIV-1 infection and/or AIDS,the method comprises the step of selecting a patient who has a reducedT-cell count of less than 375/mm³. In some embodiments of the method fortreating an HIV-1 infection and/or AIDS, the method comprises the stepof selecting a patient who has a reduced T-cell count of less than200/mm³.

B. Preventing Death Of CD4 T-Cells

The present invention provides methods for preventing death of a CD4T-cell in a population of CD4 T-cells comprising HIV-1 infected anduninfected CD4 T-cells.

The methods for preventing death of a CD4 T-cell in a population of CD4T-cells comprising HIV-1 infected and uninfected CD4 T-cells can bepracticed in vitro and in vivo. For practicing the method in vitro, CD4T-cells may be prepared as human lymphoid aggregate cultures (HLACs) asdescribed herein. In some embodiments, the method comprises the step ofcontacting a CD4 T-cell in a population of CD4 T-cells comprising HIV-1infected and uninfected CD4 T-cells with a compound described herein.Thereby the death of a CD4 T-cell in a population of CD4 T-cellscomprising HIV-1 infected and uninfected CD4 T-cells is prevented. Thesurvival of CD4 T-cells using a method of the invention can bedetermined as described herein (e.g., see, Examples).

When practicing the method in vivo, in some embodiments, the methodcomprises the steps of (a) selecting a patient having a CD4 T-cell in apopulation of CD4 T-cells comprising HIV-1 infected and uninfected CD4T-cells and (b) administering to the patient a compound describedherein. Thereby the death of a CD4 T-cell in a population of CD4 T-cellscomprising HIV-1 infected and uninfected CD4 T-cells is prevented.

In some embodiments of the method for preventing death of a CD4 T-cellin a population of CD4 T-cells comprising HIV-1 infected and uninfectedCD4 T-cells, the method comprises the step of selecting a patient whohas a reduced total lymphocyte count of less than 1,000/mm³. In otherembodiments, the method comprises the step of selecting a patient whohas a reduced total lymphocyte count of less than 750/mm³. In yet otherembodiments, the method comprises the step of selecting a patient whohas a reduced total lymphocyte count of less than 500/mm³. In someembodiments of the method for preventing death of a CD4 T-cell in apopulation of CD4 T-cells comprising HIV-1 infected and uninfected CD4T-cells, the method comprises the step of selecting a patient who has areduced T-cell count of less than 500/mm³. In other embodiments, themethod comprises the step of selecting a patient who has a reducedT-cell count of less than 375/mm³. In yet other embodiments, the methodcomprises the step of selecting a patient who has a reduced T-cell countof less than 200/mm³.

In some embodiments of this method, the CD4 T-cell comprises incompleteHIV-1 nucleic acids.

In some embodiments of this method, the method comprises the step ofcontacting the CD4 T-cell with a caspase-1 inhibitor, more specifically,with a caspase-1 inhibitor described herein.

In some embodiments of the method of preventing the death of CD4T-cells, the caspase-1 inhibitor is a caspase-1 inhibitor having Formula1a or 1b. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 2. In some embodiments, the caspase-1 inhibitoris a caspase-1 inhibitor having Formula 3. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 4, 4.1, 4.2,or 4.3. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 5. In some embodiments, the caspase-1 inhibitoris a caspase-1 inhibitor having Formula 6. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 7. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 8, 8.1, or 8.2. In some embodiments, the caspase-1 inhibitor isa caspase-1 inhibitor having Formula 9 or 9.1. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 10. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 11. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 12. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 13. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 14. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 15. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 16, 16.1, 16.2, 16.3,16.4, 16.5, 16.6, or 16.7. In some embodiments, the caspase-1 inhibitoris a caspase-1 inhibitor having Formula 17, 17.1, 17.2, 17.3, 17.4,17.5, 17.6, 17.7, 17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15,17.16, 17.17, 17.18, 17.19, 17.20, 17.21, or 17.22. In some embodiments,the caspase-1 inhibitor is a caspase-1 inhibitor having Formula 18A,18B, 18.1, or 18.2. In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 19, 19A, or 19B. In some embodiments,the caspase-1 inhibitor is a caspase-1 inhibitor having Formula 20 or20A. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 21 or 21A. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 22 or 22A. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 23(I), 23(II), or 23(III). In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 24. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 25. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 26. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 27. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 28. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 29. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 30. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 31A or 31B. In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 32. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 33. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 34. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 35. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 36. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 37. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 38. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 39. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 40. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 41. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 42. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 43. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor selected from the compounds depicted in FIGS. 30 and 31. Insome embodiments, the caspase-1 inhibitor is a compound disclosed in anyof the published EP patent applications, in any of published PCT patentapplications, in any of published U.S. patent applications, or in any ofthe granted U.S. patents disclosed herein and incorporated herein byreference in their entireties.

In some embodiments of the method of preventing the death of CD4T-cells, the caspase-1 inhibitor is a caspase-1 inhibitor having Formula1a, 1b, 2, 3, 4, 4.1, 4.2, 4.3, 5, 6, 7, 8, 8.1, 8.2, 9, 9.1, 10, 11,12, 13, 14, 15, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 17, 17.1,17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 17.10, 17.11, 17.12,17.13, 17.14, 17.15, 17.16, 17.17, 17.18, 17.19, 17.20, 17.21, 17.22,18A, 18B, 18.1, 18.2, 19, 19A, 19B, 20, 20A, 21, 21A, 22, 22A, 23(I),23(II), 23(III), 24, 25, 26, 27, 28, 29, 30, 31A, 31B, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, a caspase-1 inhibitor depicted in FIGS.30 and 31 or combinations thereof.

In some embodiments of this method, the method comprises the step ofadministering to the patient an anti HIV-1 compound, such as a HAARTcompound and as described further below.

Formulation, administration, therapeutic effective amounts and dosing ofpharmaceutical compositions useful in methods for preventing death of aCD4 T-cell in a population of CD4 T-cells comprising HIV-1 infected anduninfected CD4 T-cells using a compound of the present invention aredescribed below.

C. Inhibiting Formation of Bioactive Interleukin Beta

Mammalian interleukin-1 beta (IL-1β) plays an important role in variouspathologic processes, including chronic and acute inflammation andautoimmune diseases (Oppenheim et. al. 1986, Immunology Today, 7:45-56).IL-1β is synthesized as a cell associated precursor polypeptide(pro-IL-1β) that is unable to bind IL-1 receptors and is biologicallyinactive (Mosley et al., 1987, J Biol Chem 262:2941-2944). By inhibitingconversion of precursor IL-1β to mature IL-1β, the activity ofinterleukin-1 can be inhibited. Interleukin-1β converting enzyme (ICE),also known as caspase-1, is a protease responsible for the activation ofIL-1β (Thornberry et al., 1992, Nature 356:768; Yuan et al., 1993, Cell75:641). ICE is a substrate-specific cysteine protease that cleaves theinactive prointerleukin-1 to produce the mature IL-1.

As described herein (see Examples) it was found that in CD4 T-cells,abortive production of HIV-1 reverse transcripts, leads to theproduction and secretion of bioactive IL-1β, and ultimately to celldeath. The present invention provides methods for inhibiting theformation of bioactive IL-1β.

The methods for inhibiting the formation of bioactive interleukin-beta(IL-1β) can be practiced in vitro and in vivo. For practicing the methodin vitro, cells, preferably CD4 T-cells, secreting bioactive IL-1β maybe prepared as human lymphoid aggregate cultures (HLACs) as describedherein. In some embodiments, the method comprises the step of contactingcells secreting IL-1β with a compound described herein. Thereby theformation of bioactive IL-1β is inhibited. Formation of bioactive IL-1βcan be determined using assays described herein.

When practicing the method in vivo, in some embodiments, the methodcomprises the steps of (a) selecting a patient having cells secretingIL-1β and having an HIV-1 infection or being suspected of having anHIV-1 infection or having AIDS and (b) administering to the patient acompound described herein. Thereby the formation of bioactive IL-1β isinhibited.

In some embodiments of this method, cells secreting IL-1β compriseincomplete HIV-1 nucleic acids. In some embodiments of this method,cells secreting IL-1β are infected with HIV-1. In some embodiments ofthis method, cells secreting IL-1β comprise an HIV-1 expression vector.Suitable HIV-1 expression vectors are described herein.

In some embodiments of this method, the method comprises the step ofcontacting a cell secreting IL-1β with a caspase-1 inhibitor, morespecifically, with a caspase-1 inhibitor described herein.

In some embodiments of the method for inhibiting the formation ofbioactive interleukin-beta (IL-1β), the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 1a or 1b. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 2. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 3. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 4, 4.1, 4.2, or 4.3. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 5. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 6. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 7. In some embodiments, the caspase-1 inhibitoris a caspase-1 inhibitor having Formula 8, 8.1, or 8.2. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 9 or 9.1. In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 10. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 11. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 12. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 13. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 14. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 15. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, or16.7. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7,17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15, 17.16, 17.17,17.18, 17.19, 17.20, 17.21, or 17.22. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 18A, 18B, 18.1, or18.2. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 19, 19A, or 19B. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 20 or 20A.In some embodiments, the caspase-1 inhibitor is a caspase-1 inhibitorhaving Formula 21 or 21A. In some embodiments, the caspase-1 inhibitoris a caspase-1 inhibitor having Formula 22 or 22A. In some embodiments,the caspase-1 inhibitor is a caspase-1 inhibitor having Formula 23(I),23(II), or 23(III). In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 24. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 25. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 26. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 27. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 28. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 29. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 30. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 31A or 31B. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 32. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 33. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 34. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 35. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 36. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 37. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 38. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 39. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 40. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 41. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 42. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 43. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor selectedfrom the compounds depicted in FIGS. 30 and 31. In some embodiments, thecaspase-1 inhibitor is a compound disclosed in any of the published EPpatent applications, in any of published PCT patent applications, in anyof published U.S. patent applications, or in any of the granted U.S.patents disclosed herein and incorporated herein by reference in theirentireties.

In some embodiments of the method for inhibiting the formation ofbioactive interleukin-beta (IL-1β), the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 1a, 1b, 2, 3, 4, 4.1, 4.2, 4.3, 5, 6,7, 8, 8.1, 8.2, 9, 9.1, 10, 11, 12, 13, 14, 15, 16, 16.1, 16.2, 16.3,16.4, 16.5, 16.6, 16.7, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7,17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15, 17.16, 17.17,17.18, 17.19, 17.20, 17.21, 17.22, 18A, 18B, 18.1, 18.2, 19, 19A, 19B,20, 20A, 21, 21A, 22, 22A, 23(I), 23(II), 23(III), 24, 25, 26, 27, 28,29, 30, 31A, 31B, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, acaspase-1 inhibitor depicted in FIGS. 30 and 31 or combinations thereof.

In some embodiments of this method, the method comprises the step ofadministering to the patient an anti HIV-1 compound, such as a HAARTcompound and as described further below.

Formulation, administration, therapeutic effective amounts and dosing ofpharmaceutical compositions useful in methods for inhibiting theformation of bioactive interleukin-beta using a compound of the presentinvention are described below.

D. Inhibiting Pyroptosis

As described herein (see Examples) it was found that abortive productionof HIV-1 reverse transcripts leads to pyroptosis and ultimately to celldeath.

The present invention provides methods for inhibiting pyroptosis.

The methods for inhibiting pyroptosis can be practiced in vitro and invivo. For practicing the method in vitro, cells, preferably CD4 T-cellsundergoing pyroptosis may be prepared as human lymphoid aggregatecultures (HLACs) as described herein. In some embodiments, the methodcomprises the step of contacting cells undergoing pyroptosis with acompound described herein, thereby inhibiting pyroptosis. Inhibition ofpyroptosis can be determined using assays described herein.

When practicing the method in vivo, in some embodiments, the methodcomprises the steps of (a) selecting a patient having cells undergoingpyroptosis and having an HIV-1 infection or being suspected of having anHIV-1 infection or having AIDS and (b) administering to the patient acompound described herein. Thereby the pyroptosis is inhibited.

In some embodiments of this method, cells undergoing pyroptosis compriseincomplete HIV-1 nucleic acids. In some embodiments of this method,cells undergoing pyroptosis are infected with HIV-1. In some embodimentsof this method, cells undergoing pyroptosis comprise an HIV-1 expressionvector. Suitable HIV-1 expression vectors are described herein.

In some embodiments of this method, the method comprises the step ofcontacting a cell undergoing pyroptosis with a caspase-1 inhibitor, morespecifically, with a caspase-1 inhibitor described herein.

In some embodiments of the method for inhibiting pyroptosis, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 1a or 1b. Insome embodiments, the caspase-1 inhibitor is a caspase-1 inhibitorhaving Formula 2. In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 3. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 4, 4.1, 4.2, or 4.3.In some embodiments, the caspase-1 inhibitor is a caspase-1 inhibitorhaving Formula 5. In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 6. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 7. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 8, 8.1, or 8.2. In some embodiments, the caspase-1 inhibitor isa caspase-1 inhibitor having Formula 9 or 9.1. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 10. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 11. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 12. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 13. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 14. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 15. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 16, 16.1, 16.2, 16.3,16.4, 16.5, 16.6, or 16.7. In some embodiments, the caspase-1 inhibitoris a caspase-1 inhibitor having Formula 17, 17.1, 17.2, 17.3, 17.4,17.5, 17.6, 17.7, 17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15,17.16, 17.17, 17.18, 17.19, 17.20, 17.21, or 17.22. In some embodiments,the caspase-1 inhibitor is a caspase-1 inhibitor having Formula 18A,18B, 18.1, or 18.2. In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 19, 19A, or 19B. In some embodiments,the caspase-1 inhibitor is a caspase-1 inhibitor having Formula 20 or20A. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 21 or 21A. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 22 or 22A. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 23(I), 23(II), or 23(III). In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 24. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 25. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 26. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 27. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 28. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 29. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 30. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 31A or 31B. In some embodiments, the caspase-1 inhibitor is acaspase-1 inhibitor having Formula 32. In some embodiments, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 33. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 34. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 35. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 36. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 37. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 38. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 39. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 40. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor having Formula 41. In some embodiments, the caspase-1inhibitor is a caspase-1 inhibitor having Formula 42. In someembodiments, the caspase-1 inhibitor is a caspase-1 inhibitor havingFormula 43. In some embodiments, the caspase-1 inhibitor is a caspase-1inhibitor selected from the compounds depicted in FIGS. 30 and 31. Insome embodiments, the caspase-1 inhibitor is a compound disclosed in anyof the published EP patent applications, in any of published PCT patentapplications, in any of published U.S. patent applications, or in any ofthe granted U.S. patents disclosed herein and incorporated herein byreference in their entireties.

In some embodiments of the method for inhibiting pyroptosis, thecaspase-1 inhibitor is a caspase-1 inhibitor having Formula 1a, 1b, 2,3, 4, 4.1, 4.2, 4.3, 5, 6, 7, 8, 8.1, 8.2, 9, 9.1, 10, 11, 12, 13, 14,15, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 17, 17.1, 17.2, 17.3,17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14,17.15, 17.16, 17.17, 17.18, 17.19, 17.20, 17.21, 17.22, 18A, 18B, 18.1,18.2, 19, 19A, 19B, 20, 20A, 21, 21A, 22, 22A, 23(I), 23(II), 23(III),24, 25, 26, 27, 28, 29, 30, 31A, 31B, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, a caspase-1 inhibitor depicted in FIGS. 30 and 31 orcombinations thereof.

In some embodiments of this method, the method comprises the step ofadministering to the patient an anti HIV-1 compound, such as a HAARTcompound and as described further below.

Formulation, administration, therapeutic effective amounts and dosing ofpharmaceutical compositions useful in methods for inhibiting pyroptosisusing a compound of the present invention are described below.

E. Combination Therapy

In a preferred embodiment of the present invention, a composition of theinvention is used in a method for treating an HIV-1 infection or AIDS ina patient in need of such treatment. Preferably this method is practicedin vivo. Preferably this method is practiced in a host infected withHIV-1, e.g., a human infected with HIV-1. In some embodiments, thismethod comprises the step of administering to the HIV-1 infected host atherapeutically effective amount of a composition comprising aneffective amount of a caspase-1 inhibitor or a pharmaceuticallyacceptable salt, prodrug or active derivative of such a substance.

Importantly, unlike current antiretroviral drugs designed to interferewith viral components, the caspase-1 inhibitors, do not target HIV-1itself. Instead these inhibitors target the host CD4 T-cells themselvesand thus obviate any potential problems with viral drug resistance. Thisapproach can be used in combination with antiviral drugs, and beparticularly useful for treatment of a patient with acute inflammationassociated with rapid CD4 T-cell decline, or in a patient who hasdeveloped resistance to multiple drugs and for whom few or notherapeutic options remain.

In order to increase the effectiveness of methods for the treatment ofan HIV-1 infection and/or AIDS, it may be desirable to combine aninhibitor for caspase-1 activity with other agents effective in thetreatment or prevention of HIV-1 infection or AIDS, such as an antiHIV-1 compound. When practiced in vivo, methods of the presentinvention, optionally comprise the step of administering HAART. Thus, inyet another embodiment of the present invention, a method of treating anHIV-1 infection or AIDS in an HIV-1 infected host in vivo comprises thestep of administering highly active antiretroviral therapy (HAART). Thecurrent standard of care using HAART is usually a combination of atleast three nucleoside reverse transcriptase inhibitors and frequentlyincludes a protease inhibitors, or alternatively a non-nucleosidereverse transcriptase inhibitor. Patients who have low CD4+ cell countsor high plasma RNA levels may require more aggressive HAART. Patientswith relatively normal CD4+ cell counts and low to non-measurable levelsof plasma HIV RNA over prolonged periods (i.e. slow or non-progressors)may require less aggressive HAART. For antiretroviral-naive patients whoare treated with initial antiretroviral regimen, different combinations(or cocktails) of antiretroviral drugs can be used.

Preferably, a composition comprising an inhibitor for the activationand/or activity of caspase-1 may be coadministered with a “cocktail” ofnucleoside reverse transcriptase inhibitors, non-nucleoside HIV reversetranscriptase inhibitors, and protease inhibitors, i.e., anti HIV-1compounds. For example, a composition comprising an inhibitor for theactivation and/or activity of caspase-1 may be coadministered with acocktail of two nucleoside reverse transcriptase inhibitors (e.g.ZIDOVUDINE® (AZT) and LAMIVUDINE® (3TC)), and one protease inhibitor(e.g. INDINAVIR® (MK-639)). A composition comprising an inhibitor forthe activation and/or activity of caspase-1 may also be coadministeredwith a cocktail of one nucleoside reverse transcriptase inhibitor (e.g.STAVUDINE® (d4T)), one non-nucleoside reverse transcriptase inhibitor(e.g. NEVIRAPINE® (BI-RG-587)), and one protease inhibitor (e.g.NELFINAVIR® (AG-1343)). Alternatively, a composition comprising aninhibitor for the activation and/or activity of caspase-1 may becoadministered with a cocktail of one nucleoside reverse transcriptaseinhibitor (e.g. ZIDOVUDINE® (AZT)), and two protease inhibitors (e.g.NELFINAVIR® (AG-1343) and SAQINAVIR® (Ro-31-8959)).

In some embodiments, a composition comprising an inhibitor for theactivation and/or activity of caspase-1 may be coadministered with anHIV-1 protease inhibitor. Typical suitable protease inhibitors for usein combination therapy include saquinavir (Ro 31-8959) available in hardgel capsules (INVIRASE®) and as soft gel capsules (FORTOVASE®) fromRoche Pharmaceuticals, Nutley, N.J. 07110-1199; RITONAVIR® (ABT-538,NORVIR®) from Abbott Laboratories, Abbott Park, Ill. 60064; indinavir(MK-639, CRIXIVAN®) from Merck & Co., Inc., West Point, Pa. 19486-0004;nelfnavir (AG-1343, VIRACEPT®) from Agouron Pharmaceuticals, Inc., LaJolla Calif. 92037-1020; amprenavir (141W94, AGENERASE®), a non-peptideprotease inhibitor under development by Vertex Pharmaceuticals, Inc.,Cambridge, Mass. 02139-4211 and available from Glaxo-Wellcome, ResearchTriangle, N.C. under an expanded access program; LASINAVIR® (BMS-234475)available from Bristol-Myers Squibb, Princeton, N.J. 08543 (originallydiscovered by Novartis, Basel, Switzerland (CGP-61755); DMP-450, acyclic urea discovered by Dupont and under development by TrianglePharmaceuticals; BMS-2322623, an azapeptide under development byBristol-Myers Squibb, Princeton, N.J. 08543, as a 2nd-generation HIV-1PI; ABT-378 under development by Abbott, Abbott Park, Ill. 60064; andAG-1549 an orally active imidazole carbamate discovered by Shionogi(Shionogi #S-1153) and under development by Agouron Pharmaceuticals,Inc., LaJolla Calif. 92037-1020.

Other antiviral agents for use in combination therapy with a caspase-1include hydroxyurea, ribavirin, IL-2, IL-12, pentafuside and YissumProject No. 11607. Hydroxyurea (Droxia), an inhibitor of ribonucleosidetriphosphate reductase, the enzyme involved in the activation ofT-cells, was discovered at the NCI and is under development byBristol-Myers Squibb; in preclinical studies, it was shown to have asynergistic effect on the activity of didanosine and has been studiedwith stavudine. IL-2 is disclosed in Ajinomoto EP-0142268, TakedaEP-0176299, and Chiron U.S. Pat. Nos. RE33653, 4,530,787, 4,569,790,4,604,377, 4,748,234, 4,752,585, and 4,949,314, and is available underPROLEUKIN® (aldesleukin) from Chiron Corp., Emeryville, Calif.94608-2997 as a lyophilized powder for IV infusion or sc administrationupon reconstitution and dilution with water; a dose of about 1 to about20 million IU/day, sc is preferred; a dose of about 15 million IU/day,sc is more preferred. IL-12 is disclosed in WO96/25171 and is availablefrom Roche Pharmaceuticals, Nutley, N.J. 07110-1199 and American HomeProducts, Madison, N.J. 07940; a dose of about 0.5 microgram/kg/day toabout 10 microgram/kg/day, sc is preferred. Pentafuside (DP-178, T-20) a36-amino acid synthetic peptide, is disclosed in U.S. Pat. No. 5,464,933licensed from Duke University to Trimeris which is developingpentafuside in collaboration with Duke University; pentafuside acts byinhibiting fusion of HIV-1 to target membranes. Pentafuside (3 100mg/day) is given as a continuous sc infusion or injection together withefavirenz and 2 PI's to HIV-1 positive patients refractory to a triplecombination therapy; use of 100 mg/day is preferred. Yissum Project No.11607, a synthetic protein based on the HIV-1 Vif protein, is underpreclinical development by Yissum Research Development Co., Jerusalem91042, Israel. Ribavirin,1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, is available fromICN Pharmaceuticals, Inc., Costa Mesa, Calif.; its manufacture andformulation are described in U.S. Pat. No. 4,211,771.

In some embodiments, coadministration comprises administering to apatient (i) a caspase-1 inhibitor and (ii) an anti HIV-1 compound. Insome embodiments coadministration comprises administering to a patient(i) a caspase-1 inhibitor having Formula 1a, 1b, 2, 3, 4, 4.1, 4.2, 4.3,5, 6, 7, 8, 8.1, 8.2, 9, 9.1, 10, 11, 12, 13, 14, 15, 16, 16.1, 16.2,16.3, 16.4, 16.5, 16.6, 16.7, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6,17.7, 17.8, 17.9, 17.10, 17.11, 17.12, 17.13, 17.14, 17.15, 17.16,17.17, 17.18, 17.19, 17.20, 17.21, 17.22, 18A, 18B, 18.1, 18.2, 19, 19A,19B, 20, 20A, 21, 21A, 22, 22A, 23(I), 23(II), 23(III), 24, 25, 26, 27,28, 29, 30, 31A, 31B, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, acaspase-1 inhibitor depicted in FIGS. 30 and 31 or combinations thereofand (ii) an anti HIV-1 compound.

Coadministration in the context of this invention is defined to mean theadministration of more than one therapeutic in the course of acoordinated treatment to achieve an improved clinical outcome. Suchcoadministration may also be coextensive, that is, occurring duringoverlapping periods of time. Further discussion of such conventionaltreatment can be found in the art (e.g., Gulick, 1997; Qual Life Res6:471-474; Henry et al., 1997, Postgrad Med 102:100-107; Hicks, 1997,Radiol Clin North Am 35:995-1005; Goldschmidt, 1996, Am Fam Physician54:574-580).

V. Pharmaceutical Compositions

In one aspect the present invention provides a pharmaceuticalcomposition or a medicament comprising an inhibitor for the activationand/or activity of caspase-1 of the present invention and apharmaceutically acceptable carrier. A pharmaceutical composition ormedicament can be administered to a subject for the treatment of, forexample, a condition or disease as described herein.

A pharmaceutical composition may include any combinations of one or moreinhibitors for the activation and/or activity of caspase-1.

A. Formulation And Administration

Compounds of the present invention, such as the inhibitors for theactivation and/or activity of caspase-1 described herein, are useful inthe manufacture of a pharmaceutical composition or a medicamentcomprising an effective amount thereof in conjunction or mixture withexcipients or carriers suitable for either enteral or parenteralapplication.

As a non-limiting example, in some embodiments of the present invention,a composition comprises a peptide caspase-1 inhibitor.

As a non-limiting example, in some embodiments of the present invention,a composition comprises a non-peptide caspase-1 inhibitor.

As a non-limiting example, in some embodiments of the present invention,a composition comprises caspase-1 inhibitor having Formula 1a or 1b:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein:

next to R³ represents a single or double bond; Z is oxygen or sulfur; R¹is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y; R is a C₁₋₁₂aliphatic, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl; Y is anelectronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is a group capable of fitting into the S2 sub-siteof a caspase; R⁴ is hydrogen or a C₁₋₆ aliphatic group that isoptionally interrupted by —O—, —S—, —SO₂—, —CO—, —NH—, or —N(C₁₋₄alkyl)-, or R³ and R⁴ taken together with their intervening atomsoptionally form a 3-7 membered ring having 0-2 heteroatoms selected fromnitrogen, oxygen or sulfur; Ring A is a nitrogen-containing mono-, bi-or tricyclic ring system having 0-5 additional ring heteroatoms selectedfrom nitrogen, oxygen or sulfur; Ring B is a nitrogen-containing 5-7membered ring having 0-2 additional ring heteroatoms selected fromnitrogen, oxygen or sulfur; R⁵ is R⁶, (CH₂)_(n)R⁶, COR^(E), CO₂R⁶,SO₂R⁶, CON(R⁶)₂, or SO₂N(R⁶)₂; n is one to three; and each R⁶ isindependently selected from hydrogen, an optionally substituted C₁₋₄aliphatic group, an optionally substituted C₆₋₁₀ aryl group, or a mono-or bicyclic heteroaryl group having 5-10 ring atoms.

As a non-limiting example, in some embodiments of the present invention,a composition comprises caspase-1 inhibitor having Formula 2:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is H, R⁴, haloalkyl,CHN₂, CH₂Cl, CH₂F, —CH₂OPO(R⁴)₂, —CH₂OPO(OR⁴)₂, or —C₁₋₂alkyl-R³—R⁴; R²is a P₄—P₃—P₂, P₃—P₂, or P₂ moiety of a caspase-1 inhibitor; R³ is —O—,—NH—, —NR⁴—, —S—, or —O(C═O)—; R⁴ is C₁₋₁₂aliphatic, C₆₋₁₀aryl, 5-10membered heterocyclyl, 5-10 membered heteroaryl, C₃₋₁₀cycloaliphatic,—(C₁₋₆alkyl)-C₆₋₁₀aryl, —(C₁₋₆ alkyl)-(5-10 membered heteroaryl),—(C₁₋₆alkyl)-(5-10 membered heterocyclyl), or —(C₁₋₆alkyl)-C₃₋₁₀cycloaliphatic; wherein said R⁴ group is optionallysubstituted with 0-5 J and 0-2 J²; or two R⁴ groups, together with theatom to which they are attached, form a 3-8 membered monocyclic or 8-12membered bicyclic ring optionally substituted with 0-5 J and 0-2 J²; Jis halogen, —OR′, —NO₂, —CN, —CF₃, —OCF₃, —R′, 1,2-methylenedioxy,1,2-ethylenedioxy, —N(R′)₂, —SR′, —SOR′, SO₂R′, —SO₂N(R′)₂, —SO₃R′,C(O)R′, —C(O)C(O)R′, —C(O)C(O)OR′, —C(O)C(O)N(R′)₂, —C(O)CH₂C(O)R′,—C(S)R′, —C(S)OR′, —C(O)OR′, —OC(O)R′, —C(O)N(R′)₂, —OC(O)N(R′)₂,—C(S)N(R′)₂, —(CH₂)₀₋₂NHC(O)R′, —N(R′)N(R′)COR′, —N(R)N(R)C(O)OR′,—N(R′)N(R′)CON(R′)₂, —N(R′)SO₂R′, —N(R′)SO₂N(R′)₂, —N(R′)C(O)OR′,—N(R′)C(O)R′, —N(R′)C(S)R′, —N(R′)C(O)N(R′)₂, —N(R′)C(S)N(R′)₂,—N(COR′)COR′, —N(OR′)R′, —CN, —C(═NR′)N(R)₂, —C(O)N(OR′)R′, —C(═NOR′)R′,—OP(O)(OR′)₂, —P(O)(R′)₂, —P(O)(OR′)₂, or —P(O)(H)(OR′); J₂ is ═NR′,═N(OR′), ═O, or ═S; R′ is H, C₁₋₁₂aliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl, C₃₋₁₀ cycloaliphatic,—(C₁₋₆alkyl)-C₆₋₁₀aryl, —(C₁₋₆alkyl)-(5-10 membered heteroaryl),—(C₁₋₆alkyl)-(5-10 membered heterocyclyl), or —(C₁₋₆ alkyl)-C₃₋₁₀cycloaliphatic; each R′ is independently and optionally substituted with0-5 occurrences of H, C₁₋₆alkyl, CF₃, halogen, NO₂, OCF₃, CN, OH,O(C₁₋₆alkyl), NH₂, N(C₁₋₆alkyl), N(C₁₋₆alkyl)₂, C(═O)CH₃, or C₁₋₆alkyloptionally interrupted 1 time with a heteroatom selected from O, N, andS; wherein each C₁₋₆alkyl is unsubstituted; unless otherwise indicated,any group with suitable valence is optionally substituted with 0-5 J and0-2 J².

Benzenesulfonyl-ureas described herein are preferably destined for themanufacture of orally administrable preparations and can be applied assuch or in the form of their salts or in the presence of substancescausing salt formation. For the formation of salts there can be used:alkaline agents, for instance, alkali metal hydroxides, alkaline earthmetal hydroxides, alkali metal carbonates, alkaline earth metalcarbonates, alkali metal bicarbonates, alkaline earth metalbicarbonates, but likewise organic bases, particularly tertiary nitrogenbases, if the latter are tolerated by the host.

In some embodiments, a pharmaceutical preparation is a tablet containingin addition to a compound of the invention an adjuvant or carrier suchas talc, starch, lactose, tragacanth or magnesium stearate.

In some embodiments, a preparation containing the above-mentionedbenzenesulfonyl-ureas as active substance, for instance, a tablet or apowder, with or without the above-mentioned additions, is favorablybrought into a suitable dosage unit form. The dose chosen should complywith the activity of the benzenesulfonyl-urea used and the desiredeffect. Favorably, the dosage per unit amounts to about 0.5 to 100milligrams, preferably to 2-10 milligrams, but considerably higher orlower dosage units can likewise be used which, if desired, are dividedor multiplied prior to application.

Pharmaceutical compositions or medicaments for use in the presentinvention can be formulated by standard techniques using one or morephysiologically acceptable carriers or excipients. Suitablepharmaceutical carriers are described herein and in “Remington'sPharmaceutical Sciences” by E. W. Martin. The compounds of the presentinvention and their physiologically acceptable salts and solvates can beformulated for administration by any suitable route that achieves theirintended purpose, including via inhalation, topically, nasally, orally,parenterally, or rectally. Thus, the administration of thepharmaceutical composition may be made by intradermal, subdermal,intravenous, intramuscular, intranasal, intracerebral, intratracheal,intraarterial, intraperitoneal, intravesical, intrapleural,intracoronary or intratumoral injection, with a syringe or otherdevices. Transdermal administration is also contemplated, as areinhalation or aerosol administration. Tablets and capsules can beadministered orally, rectally or vaginally.

For oral administration, a pharmaceutical composition or a medicamentcan take the form of, for example, a tablet or a capsule prepared byconventional means with a pharmaceutically acceptable excipient.Preferred are tablets and gelatin capsules comprising the activeingredient, i.e., a small molecule compound of the present invention,together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose,mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystallinecellulose), glycine, pectin, polyacrylates and/or calcium hydrogenphosphate, calcium sulfate; (b) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt, metallic stearates,colloidal silicon dioxide, hydrogenated vegetable oil, corn starch,sodium benzoate, sodium acetate and/or polyethyleneglycol; for tabletsalso (c) binders, e.g., magnesium aluminum silicate, starch paste,gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone and/or hydroxypropyl methylcellulose; if desired(d) disintegrants, e.g., starches (e.g., potato starch or sodiumstarch), glycolate, agar, alginic acid or its sodium salt, oreffervescent mixtures; (e) wetting agents, e.g., sodium lauryl sulphate,and/or (f) absorbents, colorants, flavors and sweeteners.

Tablets may be either film coated or enteric coated according to methodsknown in the art. Liquid preparations for oral administration can takethe form of, for example, solutions, syrups, or suspensions, or they canbe presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives, forexample, suspending agents, for example, sorbitol syrup, cellulosederivatives, or hydrogenated edible fats; emulsifying agents, forexample, lecithin or acacia; non-aqueous vehicles, for example, almondoil, oily esters, ethyl alcohol, or fractionated vegetable oils; andpreservatives, for example, methyl or propyl-p-hydroxybenzoates orsorbic acid. The preparations can also contain buffer salts, flavoring,coloring, and/or sweetening agents as appropriate. If desired,preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

In some embodiments of the present invention, a tablet suitable for oraladministration comprises 0.3 to 100 milligrams, preferably 2 to 10milligrams, of a caspase-1 inhibitor.

As a non-limiting example, in some embodiments of the present invention,a tablet suitable for oral administration comprises 0.3 to 100milligrams, preferably 2 to 10 milligrams, of a caspase-1 inhibitorhaving Formula 1a or 1b:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein:

next to R³ represents a single or double bond; Z is oxygen or sulfur; R¹is hydrogen, —CHN₂, —R, —CH₂OR, —CH₂SR, or —CH₂Y; R is a C₁₋₁₂aliphatic, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl; Y is anelectronegative leaving group; R² is CO₂H, CH₂CO₂H, or esters, amides orisosteres thereof; R³ is a group capable of fitting into the S2 sub-siteof a caspase; R⁴ is hydrogen or a C₁₋₆ aliphatic group that isoptionally interrupted by —O—, —S—, —SO₂—, —CO—, —NH—, or —N(C₁₋₄alkyl)-, or R³ and R⁴ taken together with their intervening atomsoptionally form a 3-7 membered ring having 0-2 heteroatoms selected fromnitrogen, oxygen or sulfur; Ring A is a nitrogen-containing mono-, bi-or tricyclic ring system having 0-5 additional ring heteroatoms selectedfrom nitrogen, oxygen or sulfur; Ring B is a nitrogen-containing 5-7membered ring having 0-2 additional ring heteroatoms selected fromnitrogen, oxygen or sulfur; R⁵ is R⁶, (CH₂)_(n)R⁶, COR^(E), CO₂R⁶,SO₂R⁶, CON(R⁶)₂, or SO₂N(R⁶)₂; n is one to three; and each R⁶ isindependently selected from hydrogen, an optionally substituted C₁₋₄aliphatic group, an optionally substituted C₆₋₁₀ aryl group, or a mono-or bicyclic heteroaryl group having 5-10 ring atoms.

As a non-limiting example, in some embodiments of the present invention,a tablet suitable for oral administration comprises 0.3 to 100milligrams, preferably 2 to 10 milligrams, of a caspase-1 inhibitorhaving Formula 2:

or single stereoisomers, mixtures of stereoisomers, pharmaceuticallyacceptable salts or prodrugs thereof, wherein R¹ is H, R⁴, haloalkyl,CHN₂, CH₂C1, CH₂F, —CH₂OPO(R⁴)₂, —CH₂OPO(OR⁴)₂, or —C₁₋₂alkyl-R³—R⁴; R²is a P₄—P₃—P₂, P₃—P₂, or P₂ moiety of a caspase-1 inhibitor; R³ is —O—,—NH—, —NR⁴—, —S— or —O(C═O)—; R⁴ is C₁₋₁₂aliphatic, C₆₋₁₀aryl, 5-10membered heterocyclyl, 5-10 membered heteroaryl, C₃₋₁₀cycloaliphatic,—(C₁₋₆alkyl)-C₆₋₁₀aryl, (C₁₋₆ alkyl)-(5-10 membered heteroaryl),—(C₁₋₆alkyl)-(5-10 membered heterocyclyl), or —(C₁₋₆alkyl)-C₃₋₁₀cycloaliphatic; wherein said R⁴ group is optionallysubstituted with 0-5 J and 0-2 J²; or two R⁴ groups, together with theatom to which they are attached, form a 3-8 membered monocyclic or 8-12membered bicyclic ring optionally substituted with 0-5 J and 0-2 J²; Jis halogen, —OR′, —NO₂, —CN, —CF₃, —OCF₃, —R′, 1,2-methylenedioxy,1,2-ethylenedioxy, —N(R′)₂, —SR′, —SOR′, SO₂R′, —SO₂N(R′)₂, —SO₃R′,C(O)R′, —C(O)C(O)R′, —C(O)C(O)OR′, —C(O)C(O)N(R′)₂, —C(O)CH₂C(O)R′,—C(S)R′, —C(S)OR′, —C(O)OR′, —OC(O)R′, —C(O)N(R′)₂, —OC(O)N(R′)₂,—C(S)N(R′)₂, —(CH₂)₀₋₂NHC(O)R′, —N(R′)N(R′)COR′, —N(R)N(R)C(O)OR′,—N(R′)N(R′)CON(R′)₂, —N(R′)SO₂R′, —N(R′)SO₂N(R′)₂, —N(R′)C(O)OR′,—N(R′)C(O)R′, —N(R′)C(S)R′, —N(R′)C(O)N(R′)₂, —N(R′)C(S)N(R′)₂,—N(COR′)COR′, —N(OR′)R′, —CN, —C(═NR′)N(R)₂, —C(O)N(OR′)R′, —C(═NOR′)R′,—OP(O)(OR′)₂, —P(O)(R′)₂, —P(O)(OR′)₂, or —P(O)(H)(OR′); J₂ is ═NR′,═N(OR′), ═O, or ═S; R′ is H, C₁₋₁₂aliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl, C₃₋₁₀cycloaliphatic,—(C₁₋₆alkyl)-C₆₋₁₀aryl, —(C₁₋₆alkyl)-(5-10 membered heteroaryl),—(C₁₋₆alkyl)-(5-10 membered heterocyclyl), or —(C₁₋₆ alkyl)-C₃₋₁₀cycloaliphatic; each R′ is independently and optionally substituted with0-5 occurrences of H, C₁₋₆alkyl, CF₃, halogen, NO₂, OCF₃, CN, OH,O(C₁₋₆alkyl), NH₂, N(C₁₋₆alkyl), N(C₁₋₆alkyl)₂, C(═O)CH₃, or C₁₋₆alkyloptionally interrupted 1 time with a heteroatom selected from O, N, andS; wherein each C₁₋₆alkyl is unsubstituted; unless otherwise indicated,any group with suitable valence is optionally substituted with 0-5 J and0-2 J².

Compounds of the present invention can be formulated for parenteraladministration by injection, for example by bolus injection orcontinuous infusion. Formulations for injection can be presented in unitdosage form, for example, in ampoules or in multi-dose containers, withan added preservative. Injectable compositions are preferably aqueousisotonic solutions or suspensions, and suppositories are preferablyprepared from fatty emulsions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, salts for regulatingthe osmotic pressure and/or buffers. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, for example, sterile pyrogen-free water, before use. Inaddition, they may also contain other therapeutically valuablesubstances. The compositions are prepared according to conventionalmixing, granulating or coating methods, respectively, and contain about0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

For administration by inhalation, the compounds may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, forexample, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase, for example, lactose or starch.

Suitable formulations for transdermal application include an effectiveamount of a compound of the present invention with carrier. Preferredcarriers include absorbable pharmacologically acceptable solvents toassist passage through the skin of the host. For example, transdermaldevices are in the form of a bandage comprising a backing member, areservoir containing the compound optionally with carriers, optionally arate controlling barrier to deliver the compound to the skin of the hostat a controlled and predetermined rate over a prolonged period of time,and means to secure the device to the skin. Matrix transdermalformulations may also be used.

Suitable formulations for topical application, e.g., to the skin andeyes, are preferably aqueous solutions, ointments, creams or gelswell-known in the art. Such may contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

The compounds can also be formulated in rectal compositions, forexample, suppositories or retention enemas, for example, containingconventional suppository bases, for example, cocoa butter or otherglycerides.

Furthermore, the compounds can be formulated as a depot preparation.Such long-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can, for example, comprise metal or plasticfoil, for example, a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

In some embodiments of the present invention, a pharmaceuticalcomposition or medicament comprises an effective amount of an inhibitorfor the activation and/or activity of caspase-1 as described above, andanother therapeutic agent, such as a component used for HAART, asdescribed herein. When used with compounds of the invention, suchtherapeutic agent may be used individually (e.g., a component used forHAART and compounds of the present invention), sequentially (e.g., acomponent used for HAART and compounds of the present invention for aperiod of time followed by e.g., a second component used for HAART andcompounds of the present invention), or in combination with one or moreother such therapeutic agents (e.g., a reverse transcriptase inhibitorused for HAART, a protease inhibitor used for HAART, and compounds ofthe present invention). Administration may be by the same or differentroute of administration or together in the same pharmaceuticalformulation.

In a some embodiments of the present invention, a pharmaceuticalcomposition comprises (i) a caspase-1 inhibitor or a pharmaceuticallyacceptable salt, prodrug or active derivative of such a substance and(ii) a pharmaceutically acceptable carrier.

In a some embodiments of the present invention, a pharmaceuticalcomposition comprises (i) a caspase-1 inhibitor or a pharmaceuticallyacceptable salt, prodrug or active derivative of such a substance, (ii)an inhibitor for use in HAART, and (iii) a pharmaceutically acceptablecarrier.

B. Therapeutic Effective Amount and Dosing

In some embodiments of the present invention, a pharmaceuticalcomposition or medicament is administered to a subject, preferably ahuman, at a therapeutically effective dose to prevent, treat, or controla condition or disease as described herein, such as HIV-1 infection andAIDS. The pharmaceutical composition or medicament is administered to asubject in an amount sufficient to elicit an effective therapeuticresponse in the subject. An effective therapeutic response is a responsethat at least partially arrests or slows the symptoms or complicationsof the condition or disease. An amount adequate to accomplish this isdefined as “therapeutically effective dose.”

The dosage of active compounds administered is dependent on the speciesof warm-blooded animal (mammal), preferably a human, the body weight,age, individual condition, surface area of the area to be treated and onthe form of administration. The size of the dose also will be determinedby the existence, nature, and extent of any adverse effects thataccompany the administration of a particular small molecule compound ina particular subject. A unit dosage for oral administration to a mammalof about 50 to 70 kg may contain between about 5 and 500 mg of theactive ingredient. Typically, a dosage of the active compounds of thepresent invention, is a dosage that is sufficient to achieve the desiredeffect. Optimal dosing schedules can be calculated from measurements ofcompound accumulation in the body of a subject. In general, dosage maybe given once or more daily, weekly, or monthly. Persons of ordinaryskill in the art can easily determine optimum dosages, dosingmethodologies and repetition rates.

In some embodiments of the present invention, a pharmaceuticalcomposition or medicament comprising compounds of the present inventionis administered in a daily dose in the range from about 0.1 mg of eachcompound per kg of subject weight (0.1 mg/kg) to about 1 g/kg formultiple days. In other embodiments, the daily dose is a dose in therange of about 5 mg/kg to about 500 mg/kg. In yet other embodiments, thedaily dose is about 10 mg/kg to about 250 mg/kg. In other embodiments,the daily dose is about 25 mg/kg to about 150 mg/kg. A preferred dose isabout 10 mg/kg. The daily dose can be administered once per day ordivided into subdoses and administered in multiple doses, e.g., twice,three times, or four times per day. However, as will be appreciated by askilled artisan, inhibitors for the activation and/or activity ofcaspase-1 may be administered in different amounts and at differenttimes.

In some embodiments, a tablet comprises from 0.5 to 100 mg of the activeingredient of a compound described herein, preferably from 1 to 50 mg,more preferably from 1.5 to 25 mg, even more preferably from 2 to 10 mg.

To achieve the desired therapeutic effect, compounds may be administeredfor multiple days at the therapeutically effective daily dose. Thus,therapeutically effective administration of compounds to treat acondition or disease described herein in a subject requires periodic(e.g., daily) administration that continues for a period ranging fromthree days to two weeks or longer. Typically, compounds will beadministered for at least three consecutive days, often for at leastfive consecutive days, more often for at least ten, and sometimes for20, 30, 40 or more consecutive days. When used to prevent the appearanceor manifestation of a condition or disease described herein,administration of a pharmaceutical composition may be done daily for aslong as the appearance or manifestation of the condition or disease isto be prevented. While consecutive daily doses are a preferred route toachieve a therapeutically effective dose, a therapeutically beneficialeffect can be achieved even if the compounds are not administered daily,so long as the administration is repeated frequently enough to maintaina therapeutically effective concentration of the compounds in thesubject. For example, one can administer the compounds every other day,every third day, or, if higher dose ranges are employed and tolerated bythe subject, once a week. A preferred dosing schedule, for example, isadministering daily for a week, one week off and repeating this cycledosing schedule for 3-4 cycles.

Optimum dosages, toxicity, and therapeutic efficacy of compoundsdescribed herein may vary depending on the relative potency ofindividual compounds and can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, for example, bydetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and can be expressed as the ratio, LD₅₀/ED₅₀. Compounds thatexhibit large therapeutic indices are preferred. While compounds thatexhibit toxic side effects can be used, care should be taken to design adelivery system that targets such compounds to the site of affectedtissue to minimize potential damage to normal cells and, thereby, reduceside effects.

The data obtained from, for example, cell culture assays and animalstudies can be used to formulate a dosage range for use in humans. Thedosage of such small molecule compounds lies preferably within a rangeof circulating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration. For any compoundsused in the methods of the invention, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (the concentration of thetest compound that achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography (HPLC).In general, the dose equivalent of compounds is from about 1 ng/kg to100 mg/kg for a typical subject.

For the treatment of an HIV-1 infection and/or AIDS or for preventingthe death of a CD4 T-cell in a population of CD4 T-cells comprisingHIV-1 infected and uninfected CD4 T-cells there may be no fixed dosageregimen for administering a some caspase-1 inhibitor. The patient'sviral load or CD4 T-cell count may be measured periodically to determinethe minimum effective dose for the patient.

In some embodiments, a starting dose of a caspase-1 inhibitor is 2.5 to5 mg daily, which may be administered with breakfast or a first mainmeal. In some embodiments, a maintenance dose for a caspase-1 inhibitoris in the range of 1.25 to 20 mg daily, which may be given as a singledose or in divided doses. Dosage increases should be made in incrementsof no more than 2.5 mg at weekly intervals based upon the patient'sresponse (such as increasing or lowering viral load, increasing ordecreasing CD4 T-cell count). Once-a-day therapy is usuallysatisfactory, based upon usual meal patterns and the half-life of somecaspase-1 inhibitors, some patients, particularly those receiving morethan 10 mg daily, may have a more satisfactory response with twice-a-daydosage.

In some embodiments, a starting dose of a caspase-1 inhibitor is 1 to 2mg daily, which may be administered with breakfast or a first main meal.In some embodiments, a maintenance dose for the caspase-1 inhibitor isin the range of 1 to 4 mg daily, which may be given as a single dose orin divided doses. In some embodiments, the maximum recommended dose is 8mg once daily. After reaching a dose of 2 mg, dosage increases should bemade in increments of no more than 2 mg at 1-2 week intervals based uponthe patient's response (such as increasing or lowering viral load,increasing or decreasing CD4 T-cell count). Once-a-day therapy isusually satisfactory, based upon usual meal patterns and the half-lifeof some caspase-1 inhibitors, some patients, particularly thosereceiving more than 8 mg daily, may have a more satisfactory responsewith twice-a-day dosage.

Following successful treatment, it may be desirable to have the subjectundergo maintenance therapy to prevent the recurrence of the conditionor disease treated.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. When thesymptoms have been alleviated to the desired level, treatment shouldcease. Patients may, however, require intermittent treatment on along-term basis upon any recurrence or disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of the disease,and the patient's disposition to the disease and the judgment of thetreating physician.

VI. Kits

For use in diagnostic, research, and therapeutic applications suggestedabove, kits are also provided by the invention. In the diagnostic andresearch applications such kits may include any or all of the following:assay reagents, buffers, a compound of the present invention, acaspase-1 polypeptide, an IL-1β polypeptide, an HIV-1 polypeptide, acaspase-1 nucleic acid, an IL-1β nucleic acid, an HIV-1 nucleic acid, ananti-HIV-1 polypeptide antibody, hybridization probes and/or PCRprimers, expression constructs for e.g., a virion, a cell expressing acaspase-1 polypeptide, a cell expressing an HIV-1 polypeptide, acomponent for use in HAART. A therapeutic product may include sterilesaline or another pharmaceutically acceptable emulsion and suspensionbase.

In some embodiments of the present invention, a kit comprises one ormore inhibitors for caspase-1. Optionally, the kit includes one or morecomponents used for HAART as described herein. Typically, thesecompounds are provided in a container.

This invention provides kits for use in the methods described herein. Insome embodiments of the present invention this kit comprises (i) a firstcontainer containing an inhibitor for caspase-1 and (ii) an instructionfor using the inhibitor for caspase-1 in a method of the presentinvention. In other embodiments, this kit comprises any of the compoundsdescribed herein and above, which will be provided in a separatecontainer.

In addition, a kit may include instructional materials containingdirections (i.e., protocols) for the practice of methods of thisinvention. The instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Whilethe instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials. Optionally,the instruction comprises warnings of possible side effects anddrug-drug or drug-food interactions.

A wide variety of kits and components can be prepared according to thepresent invention, depending upon the intended user of the kit and theparticular needs of the user.

In some embodiments of the present invention, the kit is apharmaceutical kit and comprises a pharmaceutical composition comprising(i) one or more inhibitors for caspase-1 and (ii) a pharmaceuticalacceptable carrier. In other embodiments, the pharmaceutical kitcomprises a component for use in HAART as described herein.Pharmaceutical kits optionally comprise an instruction stating that thepharmaceutical composition can or should be used for treating acondition or disease described herein.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

As can be appreciated from the disclosure above, the present inventionhas a wide variety of applications. While each of the elements of thepresent invention is described herein as containing multipleembodiments, it should be understood that, unless indicated otherwise,each of the embodiments of a given element of the present invention iscapable of being used with each of the embodiments of the other elementsof the present invention and each such use is intended to form adistinct embodiment of the present invention. The invention is furtherillustrated by the following examples, which are only illustrative andare not intended to limit the definition and scope of the invention inany way.

VII. Examples

The below examples are meant to illustrate specific embodiments of themethods and compositions described herein and should not be construed aslimiting the scope of the invention in any way.

Example 1 General

i. Culture and Infection of HLACs

Human tonsil or splenic tissues from routine tonsillectomies wereobtained from the National Disease Research Interchange and theCooperative Human Tissue Network and processed as previously described(Jekle et al., 2003, J Virol 77:5846-5854). In brief, tonsils or spleenwere minced, passed through a 40-μm cell strainer, and cultured in96-well U-bottomed polystyrene plates (2×10⁶ cells/well) in medium (200μl/well) consisting of RPMI 1640 supplemented with 15% heat-inactivatedfetal bovine serum, 100 μg/ml gentamicin, 200 μg/ml ampicillin, 1 mMsodium pyruvate, 1% nonessential amino acids (Mediatech), 2 mML-glutamine, and 1% fungizone (Invitrogen). All HIV-1 infections werecarried out with 20-50 ng of HIV-1 p24^(gag). Cells were incubated withthe virus for 12-16 h, washed extensively, and supplemented with freshmedium. After day 5, infections were monitored by measuring p24gaglevels in the culture medium using a FLAQ assay (Hayden et al., 2003,AIDS 17:629-631). Infected and uninfected cultures were manipulated asdescribed for individual experiments. In “indirect killing” assays,unless otherwise stated, drugs were used at the followingconcentrations: 5 μM AZT; 250 nM AMD3100; 10 μg/ml T20; 100 nMEfavirenz; 1 μM Nevirapine; 30 μM Raltegravir; 60 μM 118-D-24; 5 μMAmprenavir; 5 μM Saquinavir; 5 μM Indinavir. CFSE-labeled cells wereactivated with PHA (5 μg/ml) and IL-2 (20 ng/ml) 2 days before mixingwith effector cells.

ii. HIV-1 Constructs and Preparation of HIV-1 Virions

To generate replication-competent viruses, pNL4-3, pNLENG1 (David N.Levy, The University of Alabama, Birmingham), WEAU-16, and mutantpNL4-3: ΔVif NL4-3 (also known as pNL-ND) (Adachi et al., 1991, ArchVirol 117:45-58; Sakai et al., 1993, J Virol 67:1663-1666), TR712, SIM,GIA, GIA-SKY, SKY, pNL4-3 Δenv (an HIV molecular clone lacking the Envgene, constructed by blunting at the NheI site present in the envelopecoding region of NL4-3), E478Q (Smith et al., 1999, J Virol73:6573-6581), and pDB653 (Guo et al., 2000, J Virol 74:8980-8988)proviral expression DNA were transfected into 293T-cells by the calciumphosphate method. The GIA-SKY DNA was co-transfected with a VSV-Genvelope DNA (4:1). The medium was replaced after 16 hours. After 48hours, the supernatants were collected and clarified by sedimentation,and virions were concentrated by ultracentrifugation, and stored at −80°C. in RPMI 1640 containing 50% fetal bovine serum. All viral stocks werequantitated by measuring p24gag levels by FLAQ assay.

HIV-1 viruses were generated by transfection of proviral DNA into293T-cells by the calcium phosphate method. Virion-based fusion assaywas performed as previously described (Cavrois et al., 2002, NatBiotechnol 20:1151-1154).

iii. FACS Analysis and Gating Strategy

Uninfected cells (5×10⁶/ml) were labeled with 1 μM CFSE (MolecularProbes C1157), treated with 5 μM AZT for 24 h, and co-cultured witheffector cells. CFSE readily diffuses into cells, where intracellularesterases cleave the acetate groups, converting it to a fluorescent,membrane-impermeable dye. This dye is not transferred to adjacent cellsand does not affect cellular function. To quantify indirect killing,mixed (CFSE-labeled+effector) cultures were washed in FACS buffer (PBSsupplemented with 2 mM EDTA and 2% fetal bovine serum), stained withPE-conjugated anti-CD4, PerCP-conjugated anti-CD19, and APC-conjugatedanti-CD8 (all from BD Pharmingen) and fixed in 1% paraformaldehyde. Todetermine the absolute numbers of the viable CFSE-labeled cells, astandard number of fluorescent beads (Flow-Count Fluorospheres, BeckmanCoulter) was added to each cell-suspension sample before dataacquisition. Data were collected on a FACS Calibur (BD Biosciences) andanalyzed with Flowjo software (Treestar). The level of indirect killingwas defined by sequential gating beginning with forward scatter versusside scatter to select live lymphocytes, subgating on the CFSE-positivepopulation, and calculating the numbers of CD4 or CD8 T or B cells,divided by the number of fluorescent beads acquired.

iv. Preparation of Primary WEAU 16-8 HIV-1 Clone

Peripheral blood mononuclear cells (PBMCs) obtained 15 days after theonset of symptoms were used to isolate a molecular proviral clone ofHIV-1 (WEAU1.60). A panel of plasma-derived HIV-1 envelopes obtained atvarious times over the course of disease in this patient was alsoprepared (Wei et al., 2003, Nature 422:307-312). These envelopes weresubcloned into the WEAU1.60 backbone to create isogenic WEAU molecularclones. Each of these viruses replicated efficiently in primary PBMCcultures (data not shown). To create the proviral clone WEAU1.60, PBMCsobtained patient WEAU (Clark et al., 1991, N Engl J Med 324:954-960) 15days after the onset of infection were co-cultured withPHA/IL2-stimulated lymphocytes for 14 days and then with an H9 T-cellline for 14 days. The proviral WEAU1.60 was subcloned into pTOPO-XL, andthe flanking cellular DNA sequences were removed. Envelope 16-8,isolated 16 days after the onset of symptoms, and a panel of otherenvelopes derived from patient WEAU at sequential plasma time-points(Wei et al., 2003, Nature 422:307-312) were individually subcloned intothe pTOPO-XL WEAU1.60 plasmid using two NdeI sites located at positions6399 and 8813.

v. Virion-Based Fusion Assay

The virion-based fusion assay is performed in three successive steps:(1) incubation of target cells with virions, (2) loading of target cellswith the CCF2/AM dye, and (3) development and detection of the BlaMreaction. SupT1 cells (5×10⁵) were incubated with BlaM-Vpr containingvirions (400 ng of p24^(gag) at 37° C. for 2 hours, washed inCO₂-independent medium (GibCo BRL), and loaded with CCF2/AM dye asdescribed by the manufacturer (Invitrogen). Briefly, 2 μl of CCF2/AM (1mM) was mixed with 8 μl of 0.1% acetic acid containing 100 mg/mlPluronic-F127R and 1 ml of DEEM to constitute the loading solution.Cells were incubated in 100 μl of loading solution for 1 hour at roomtemperature. After two washes with DEEM, the BlaM reaction was executedfor 16 hours at room temperature in 200 μl of DMEM supplemented with 10%FBS and 2.5 mM probenecid, a nonspecific inhibitor of anion transport(Sigma Pharmaceuticals). Finally, the cells were washed once in DMEM andfixed in a 2% solution of paraformaldehyde. The change in emissionfluorescence of CCF2 after cleavage by the BlaM-Vpr chimera wasmonitored by flow cytometry with LSR2 (Becton Dickinson, San Jose,Calif.). Data were collected with DiVa software and analyzed with FlowJosoftware (Treestar, San Carlos, Calif.).

vi. Spinoculation

Fresh HLAC cells are cultured overnight in a V-bottom 96-well plate(1×10⁶ cells/well) in the presence of drugs and then chilled on ice.HIV-1 (600-800 ng p24gag/200 μl) is added to each well and mixed withcold cells. The cold temperature allows virion attachment but preventsvirion fusion. HIV-coated cells are then tightly packed into a pellet byhigh-speed centrifugation (1200 g) for 2 hours at 4° C. This steppresses the virus between cell membranes, exerting a uniform andconsistent attachment. Immediately after centrifugation cells arecultured at 37° C. as a pellet. This step promotes high-level attachmentof virions to target cell membranes. Immediately after centrifugation,cells are cultured at 37° C. as a pellet. This step facilitates acoordinated fusion of the attached viruses, generating a pulse of virionentry into the target cells. After 3 days of incubation, the cells aresubjected to analysis by flow cytometry.

vii. Isolation of CD4 T-Cells and Taqman-Based QPCR Analysis ofHIV-1-Infected CD4 T-Cells

Fresh HLACs were washed once with PBS and resuspended with PBScontaining 5 mM EDTA and 2% fetal bovine serum. CD4 T-cells wereisolated from HLACs by positive selection on CD4 microbeads (Miltenyi)and cultured overnight in 96-well U-bottomed plates (1×10⁶ cells/well)as described above, and AZT was added to the indicated cell samples.Next day, the rest of the drugs were added to the indicated cell samplesat concentrations as described above. Cells were chilled on ice for 15min and DNAsetreated (Ambion, 60 U/ml, 1 hour, 37° C.) NL4-3 or ΔvifNL4-3 virions (200 ng/well) were added. After a 1 hour incubation withvirions on ice, cells were spinoculated at 1200 g for 2 hours at 4° C.,incubated at 37° C. for 2 hours, washed three times with cold PBS, andresuspended in fresh RPMI with the indicated drugs. Eight or 16 hoursafter spinoculation, cell pellets were frozen at 80° C. Total DNA waspurified from each cell pellet with a DNAse kit (Qiagen). Primer andprobe sequences used to detect reverse transcription products areprovided herein. QPCR reactions were performed in triplicates in TaqManuniversal PCR master mix using each primer at 3.75 μM and probe at 2.5μM. After 15 minutes at 95° C., reactions underwent 50 cycles of 15 secat 95° C. followed by 1 min at 60° C. in an ABI Prism 7900HT (AppliedBiosystems).

viii. ISRE-GFP H35 Reporter Cells, Microscopy, and Generation ofSynthetic HIV-1 Reverse Transcription Intermediates

Hepatocyte-derived reporter ISRE-GFP H35 cells were maintained aspreviously described (King et al., 2007, Lab Chip 7:77-85; Patel et al.,2009, Proc Natl Acad Sci USA 106:12867-12872). For microscopic imaging,ISRE-GFP reporter H35 cells were cultured on 35 mm glass bottom culturedishes (MatTeck) and were treated with AZT (5 μM), Efavirenz (100 nM),or Raltegravir (30 μM) 12 hours before infection. Cells were theninfected with 2 μg/ml of replication competent VSV-G pseudotyped NL4-3.After 48 hours incubation at 37° C., cells were fixed in 2%paraformaldehyde at 25° C. for 1 hour, washed with PBS and stained with10 μg/ml Hoechst 33342 (Invitrogen) and 1 μg/ml 7-AAD (eBioscience) for15 min at 25° C. Cells were washed 3 times with PBS and were analyzedwith an Axio observer Z1 microscope (Zeiss) equipped with an EC PlanNEOFLUAR 10×/0.3 PHM27 objective, filter sets 38HE, 45, and 49, and anAxiocam MRM REV 3.

For generation of synthetic HIV-1 reverse transcription intermediates,PCR products of 150 bp, 500 bp, 1,500 bp, and 3,300 bp were generatedusing the same reverse primer: 5′-CAGTACAGGCAAAAAGCAGCTGCTTATATG-3′ (SEQID NO: 21). The following forward primers were used to amplify thecorresponding PCR product: 150-5′-GCATCCGGAGTACTTCAAGAACTGCTGAC-3′ (SEQID NO: 22); 500-5′-AAGGCAGCTGTAGATCTTAGCC-3′ (SEQ ID NO: 23);1,500-5′-ACTGCTGTGCCTTGGAATGCTAGTTGGAG-3′ (SEQ ID NO: 24);3,300-5′-ATGAGAGTGAAGGAGAAGTATCAGCACTTGTGG-3′ (SEQ ID NO:25). PCRproducts were gel purified to ensure no primer carryover. To generatessDNA, PCR products were heated at 95° C. for 5-10 minutes followed by10 minutes on ice. To generate HIV-1 pregenomic mRNA, MS HIV-1 RNAcontaining exons 1, 5, and 7 was cloned into pSP64 Poly(A) (Promega,Madison, Wis., United States). Uncapped or capped HIV-1 mRNA wasproduced by in vitro transcription with a MEGAscript™ SP6 transcriptionkit or mMESSAGE mMACHINE® SP6 Kit respectively (Ambion) according tomanufacturer's protocol. Full-length polyadenylated transcripts wereisolated using an Oligotex mRNA mini kit (Qiagen). Capped RNA wasincubated with equimolar amounts of the following primers to mimic thestrong stop DNA:RNA hybrid:5′-CTGCTAGAGATTTTCCACACTGACTAAAAGGGTCTGAGGGATCTCTAGTTACCAGAGTACCACAACAGACGGGCAGAGACTACTTTGAGCACTCAAGGCA-3′ (SEQ ID NO:26) and5′-AGCTTTATTGAGGCTTAAGCAGTGGGTTCCCTAGTTAGCCAGAGAGCTCCCAGGCTCAGATCTGGTCTAACCAGAGAGACC-3′ (SEQ ID NO: 27). To generate heteroduplexRNA, uncapped HIV-1 RNA was incubated with equimolar amounts of thefollowing primers:5′-GGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCCCTGGAAAGTCC-3′ (SEQ ID NO:28); 5′-CCTCCACTCTAACACTTCTCTCTCAGGGTCATCCATTCCATGCAGGCTCACAGGG-3′ (SEQID NO:29); 5′-GGCTCAACTGGTACTAGCTTGTAGCACCATCCAAAGGTCAGTGGATATCTGACCC3′(SEQ ID NO: 30); 5′-GCCAATCAGGGAAGTAGCCTTGTGTGTGGTAGATCCACAGATCAAGG-3′(SEQ ID NO: 31);5′-GGGAGTGAATTAGCCCTTCCAGTCCCCCCTTTTCTTTTAAAAAGTGGCTAAG-3′ (SEQ ID NO:32); 5′-GGTGTGACTGGAAAACCCACCTCTTCCTCCTCTTGTGCTTCTAGCCAGGC-3′ (SEQ IDNO: 33); 5′-GCATTGTTAGCTGCTGTATTGCTACTTGTGATTGCTCCATGTTTTTCTAGG-3′ (SEQID NO:49); 5′-CCCCATCTGCTGCTGGCTCAGCTCGTCTCATTCTTTCCCTTACAGCAGGCCATCC-3′(SEQ ID NO:34);5′-CCACTTGCCACCCATCTTATAGCAAAATCCTTTCCAAGCCCTGTCTTATTC-3′ (SEQ ID NO:35); 5′-GGCGAATAGCTCTATAAGCTGCTTGTAATACTTCTATAACCCTATACTGTCCCC-3′ (SEQID NO: 36). To generate RNA:DNA hybrids, RNA and DNA were mixed togetherat equimolar amounts, incubated at 95° C. for 5 minutes and allowed tocool to room temperature. For transfection experiments H35 ISRE-GFPcells were plated at a density of 75,000 cells/well in 12 well platesthe night before transfection. Cells were transfected with 1 μg of DNA,RNA, or RNA:DNA hybrid with Fugene (Roche) at a ratio of 3:1. 24 hoursafter transfection cells were harvested and analyzed by flow cytometryfor GFP fluorescence. All transfections were performed in triplicate andresults are representative of at least three independent experiments. Aschematic illustration of the synthetic reverse transcriptionintermediates is provided in FIG. 14E.

ix. Assays of Intracellular Cytokines and Caspases

HLACs were subjected to intracellular analysis of caspases activity andcytokine expression 3 days after spinoculation with NL4-3. Intracellularcaspase-1, -3, -6, -8, and -9 activities were analyzed with a CaspaLux1E1D2, Phiphilux G1D2, CaspaLux6 J1D2, CaspaLux 8 L1D2, and CaspaLux 9M1D2 kits (OncoImmunin), respectively, at 37° C. for 15 min, followed bystaining with PE anti-CD4, and APC anti-CD8, at 4° C. for 15 min. Forannexin V analysis, cells were stained with APC-conjugated annexin Vtogether with PE anti-CD4, FITC anti-CD8, and with 0.5 μg/ml ethidiummonoazide (E-1374, Molecular Probes) for 15 min at 4° C. Forintracellular cytokine capture, cells were incubated with 2 μM proteintransport inhibitor GolgiStop (BD Biosciences) containing monensin, and5 μg/ml Brefeldin A (EMD biosciences) at 37° C. for 6 hours beforeanalysis. Cells were then stained with anti-CD4 and anti-CD8 antibodiesat 4° C. for 15 min, and fixed in 1% paraformaldehyde at 4° C. for 1hour. Fixed cells were washed extensively with PBS and were stained in0.2% saponin buffer (PBS+2% FBS+0.2% saponin) containing PE anti-humanTNFα (R&D Systems), FITC anti-human IFN-β (PBL Biomedical laboratories),PE anti-human IL-1β (BD Biosciences), or APC anti-phosphorylated p53 atserine 37 (pS37) at 4° C. for 1 hour.

x. Protein Analysis of IL-β Maturation and Secretion

For stimulating the processing and secretion of IL-1, CD4 T-cells wereisolated from HLACs by positive selection and treated with 0.5 μM PMA(Phorbol-12-myristate-12-acetate, Calbiochem Cat. #524400) for 6 hoursat 37° C. PMA induces large intracellular stores of the 35 kDapro-IL-1β. Cells were then washed with PBS and treated with 10 μMnigericin (Sigma, Cat. # N7143) overnight at 37° C. The potassiumionophore nigericin mediates an elecroneutral exchange of intracellularK⁺ ions for extracellular protons, providing a second inflammatorystimulus, which results in the maturation and release of the bioactive17 kDa IL-1β

(Perregaux et al., 1992, J Immunol 149:1294-1303; Perregaux and Gabel,1994, J Biol Chem 269:15195-15203).

For assessing the processing and secretion of IL-1β in abortivelyinfected CD4 T-cells, CD4 T-cells were isolated from HLACs as describedabove, and were spinoculated with or without NL4-3 with the indicateddrugs as describes above in vi (Spinoculation) and in FIG. 7B. Forintracellular protein analysis, cells were lysed 72 hours afterspinoculation in RIPA buffer (150 mM NaCl, 1% Nonidet P-40 (vol/vol),0.5% AB-deoxycholate (vol/vol), 0.1% SDS (vol/vol), 50 mM Tris-HCl (pH8), 1 mM DTT), and EDTA-free Protease Inhibitor (Roche Applied Science,Cat. #04 693 132 001). For analysis of secreted IL-1β, supernatants (200μl/1 million cells) from the rest of the CD4 T-cells were collected fivedays after spinoculation and analyzed using Western blot. For Westernblots we used the Bio-Rad Criterion 15% pre-cast Tris-HCl gels. Primaryantibodies used were 1/1000 of the mouse monoclonal anti-human IL-1β(R&D Systems, Cat. # MAB201) and 1/10000 of the mouse monoclonalanti-β-Actin (Sigma, Cat. # A5316).

xi. Caspase-1 and Caspase-3 Enzyme Assays

Several assays for caspase inhibition are known in the art (e.g.,WO2001/42216) and described herein.

Assays for caspase inhibition can be based on the cleavage of afluorogenic substrate by recombinant, purified human caspase-1 orcaspase-3. The assays are run in essentially the same way as thosereported by Garcia-Calvo et al. (1998, J Biol Chem 273:32608-32613),using a substrate specific for each enzyme. The substrate for caspase-1is Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (SEQ ID NO: 37). Thesubstrate for caspase-3 is Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin(SEQ ID NO: 38). The observed rate of enzyme inactivation at aparticular inhibitor concentration, k_(obs), is computed by direct fitsof the data to the equation derived by Thornberry et al. (1994,Biochemistry 33:3943-3939) using a nonlinear least-squares analysiscomputer program (PRISM 2.0; GraphPad software). To obtain the secondorder rate constant, k_(inact), k_(obs) values are plotted against theirrespective inhibitor concentrations and k_(inact) values aresubsequently calculated by computerized linear regression.

The effectiveness of compounds against the activity of human recombinantcaspase-1 (BIOMOL Research Laboratories, Inc.) can also be measuredusing fluorescent based assays. 3 nM active enzyme are added to testcompounds dissolved in DMSO (at various concentrations) and incubated atroom temperature for 30 minutes. The tetrapeptide substrate(Ac-Trp-Glu-His-Asp-AFC, Alexis Biochemicals) is added to a finalconcentration of 4 μM to initiate the reaction, bringing the finalreaction volume to 50 μL. Preferred caspase-1 reaction buffer contains25 mM HEPES pH 7.4, 0.1% CHAPS, 50 mM KCl and 5 mM β-mercaptoethanol(β-ME). Caspase activity is monitored using Molecular Devices'Microplate Spectrofluorometer Gemini XS over 15-minutes at roomtemperature. IC₅₀ values are calculated using direct fits of the data toa 4-parameter fit using the computer application SOFTmax PRO.K_(i(apparent)) values were calculated according to Kuzmic et al. (2000,Analytical Biochem 286:45-50).

The efficacy of caspase-3 inhibitors at the cellular level can also betested in live Hela cells by the determining ability of compounds toinhibit the proteolytic cleavage of PARP (poly ADP-ribose polymerase).Briefly, in this assay, Hela cells are seeded in 96 well plates andincubated for 4 hours with staurosporine, a well characterized inducerof apoptosis, alone or together with different concentrations of acompound (e.g., 50, 25, 10 and 3 μM). After formaldehyde-based fixation,the cells are stained with a fluorescein-labeled anti-cleaved PARPantibody (Cell signaling, Cat#: 9547) and counterstained with Hoechst33342 (Invitrogen, Cat#: H3570) to mark all nuclei. Fluorescence imagesare taken on a Cellomics™ microscope system (Thermo Scientific,Pittsburgh, USA) with the Hoechst stain in the blue channel and thecleaved PARP antibody stain in the green channel. The percentage ofcleaved PARP positive cells is determined by calculating the ratiobetween nuclei with a cleaved PARP antibody staining above a certainthreshold and all (Hoechst positive) nuclei. The efficacy of caspase-3inhibition is determined by calculating the ratio between cleaved PARPpositive cells after staurosporine incubation together with compoundsand staurosporine incubation without compounds.

xii. Inhibition of IL-1β Secretion Assay

Processing of pre-IL-1β by caspase-1 can be measured in cell cultureusing a variety of cell sources. Human PBMC obtained from healthy donorsprovide a mixed population of lymphocyte and mononuclear cells thatproduce a spectrum of interleukins and cytokines in response to manyclasses of physiological stimulators.

Experimental Procedure: A test compound is dissolved in dimethylsulfoxide (DMSO,Sigma #D-2650) to give a 100 mM stock solution. This isdiluted in complete medium consisting of RPMI containing 10% heatinactivated FCS (Gibco BRL #10099-141), 2 mM L-Glutamine (Sigma,#G-7513), 100 U penicillin and 100 μg/ml streptomycin (Sigma #P-7539).The final concentration range of the test compound is adjusted from 100μM down to 6 nM over eight dilution steps. The highest concentration oftest compound is equivalent to 0.1% DMSO in the assay. Human PBMC areisolated from Buffy Coats obtained from the blood bank usingcentrifugation on Ficoll-Paque leukocyte separation medium (Amersham,#17-1440-O₂) and the cellular assay is performed in a sterile 96 wellflat-bottomed plate (Nunc). Each well contains 100 μl of the cellsuspension, 1×10⁵ cells, 50 μl of compound dilutions and 50 μl of LPS(Sigma #L-3012) at 50 ng/ml final concentration. Controls consist ofcells +/−LPS stimulation and a serial dilution of DMSO diluted in thesame way as compound. The plates are incubated for 16-18 h at 37° C. in5% CO₂ & 95% humidity atmosphere. After 16-18 h, the supernatants areharvested after centrifuging the plates at 100×g at 18° C. for 15 minand assayed for their IL-1β content. Measurement of mature IL-1β in thesupernatant is performed using the Quantikine kits (R&D Systems)according to manufacturer's instructions. Mature IL-1β levels of about600-1500 pg/ml are observed for PBMCs in positive control wells. Theinhibitory potency of the compounds can be represented by an IC₅₀ value,which is the concentration of inhibitor at which 50% of the mature IL-1βis detected in the supernatant as compared to the positive controls.

xiii. ELISA for IL-1β

Quantikine kits (R&D Systems) may be used for the measurement of matureIL-1β. Assays are performed according to the manufacturer's directions.Mature IL-1β levels of about 1-3 ng/ml in both PBMC and adherentmononuclear cell positive controls are observed. ELISA assays areperformed on 1:5, 1:10 and 1:20 dilutions of supernatants fromLPS-positive controls to select the optimal dilution for supernatants inthe test panel. The inhibitory potency of compounds can be representedby an IC₅₀ value, which is the concentration of inhibitor at which 50%of mature IL-1β is detected in the supernatant as compared to thepositive controls.

Suitable antibodies for measuring IL-1β levels by ELISA, include, butare not limited to MAB601 (R&D Systems, Inc.; monoclonal anti-humanIL-1β antibody) and BAF201 (R&D Systems, Inc.; biotinylated anti-humanIL-1β antibody).

xiv. Caspase 1 Inhibitors

Caspase 1 inhibitors, Z-YVAD-FMK (SEQ ID NO: 16) (Catalog Number FMK005)and Z-WEHD-FMK (SEQ ID NO: 13) (Catalog Number FMK002), Z-VAD-FMK(Catalog Number FMK001), Z-DEVD-FMK (SEQ ID NO: 18) (Catalog NumberFMK004), and caspase inhibitor control Z-FA-FMK (Catalog Number FMKC01)used in the examples described herein were obtained from R&D Systems,Inc. (USA).

Example 2 Selective Depletion of CD4 T-Cells by X4-Tropic HIV-1

To explore depletion of CD4 T-cells by HIV-1, HLACs made from freshlydissected human tonsillar tissues were infected with a GFP reportervirus (NLENG1), prepared from the X4-tropic NL4-3 strain of HIV-1. Thisreporter produces fully replication-competent viruses. An IRES insertedupstream of the Nef gene preserves Nef expression and supportsLTR-driven GFP expression (Levy et al., 2004, Proc Natl Acad Sci USA101:4204-4209), allowing simultaneous quantification of the dynamics ofHIV-1 infection and T-cell depletion. NL4-3 was selected becausetonsillar tissue contains a high percentage of CD4 T-cells expressingCXCR4 (90-100%). Productively infected GFP-positive cells appeared insmall numbers 3 days after infection, peaked on days 6-9, and decreaseduntil day 12, when few CD4 T-cells remained in the culture (FIG. 1)Fluorescence-linked antigen quantification (FLAQ) assay of HIV-1 p24(Hayden et al., 2003, AIDS 17:629-631) confirmed the accumulation ofviral particles in the medium between day 3 and days 8-9, when a plateauwas reached (data not shown). Interestingly, when HIV-1 p24 levelsplateaued no more than 1.5% of all cells (about 5% of CD4 T-cells) wereGFP-positive. However, although the number of CD4 T-cells was notmarkedly altered in infected cultures through six days, the culture wasalmost completely devoid of CD4 T-cells by day 9. CD8 T-cells were notdepleted in infected cultures, and CD4 T-cells were not depleted inuninfected cultures. These findings reveal marked and selectivedepletion of CD4 T-cells in HLAC cultures. However, due to the nature ofthe assay, it could not have been definitely concluded whether theprincipal mechanism of depletion involved direct or indirect effects ofHIV-1.

Example 3 Extensive Depletion of Non-Productively Infected CD4 T-Cellsin HLACs

To determine if indirect killing (formerly indicated as “bystander”) ofCD4 T-cells accounted for most of the observed cellular depletion, anexperimental strategy (Jekle et al., 2003, J Virol 77:5846-5854) wasemployed that unambiguously distinguishes between the death ofproductively and non-productively infected cells (FIG. 2A). After 6 daysof co-culture, survival analysis of CFSE-labeled cells by flow cytometry(FIG. 2B) showed extensive depletion of CD4 T-cells in cultures mixedwith HIV-infected cells but not in those mixed with uninfected cells(FIG. 2C). The relative proportion of CD8 T-cells was not altered.CD3⁺/CD8T-cells were similarly depleted, indicating that the loss wasnot an artifact of downregulated surface expression of CD4 followingdirect infection. Loss of CFSE-labeled CD4 T-cells was prevented byAMD3100, which blocks the engagement of gp120 with CXCR4, but not by thereverse transcriptase inhibitor AZT (AZT prevents productive infectionof target cells without affecting viral output from productivelyinfected cells). Thus, productive viral replication is not required forCD4 T-cell death.

To estimate the absolute numbers of all CFSE-labeled cell subsets, astandard number of fluorescent beads was added to the cell suspensions(FIG. 2D). In contrast to the sharp decline in CD4 T-cells, the absolutenumbers of CD8 T and B-cells were unaltered. Separating the HLAC intodistinct cell types revealed that cell death occurred in purifiedpopulations of CD4 T-cells suggesting that other cell types did notmediate the killing. (FIG. 3). In all instances, CD4-specific killingwas prevented by AMD3100 but not AZT. Importantly, the extent of CD4T-cell depletion in the presence of AZT was similar to that observedwhen no antiviral drugs were added (FIG. 2C and FIG. 1, respectively).Together, these results suggest that indirect killing is the predominantmechanism for CD4 T-cell depletion in HIV-infected HLACs.

Example 4 Indirect Killing is an Intrinsic Property of CD4 T-Cells

It was investigated whether indirect killing of CD4 T-cells in HLACsrequires the presence of other cell types. To this end, CD4 T-cells, CD8T-cells, and B cells from complete HLACs were isolated and assessed forthe level of indirect killing occurring in single and combined culturesas indicated (FIG. 3A). Interestingly, indirect killing was equallyeffective in co-cultures containing only CD4 T-cells as found incultures containing additional CD8 T and B cells (FIG. 3B), suggestingthat interactions between CD4 T-cells are sufficient to induce the celldeath response. The somewhat greater depletion of CD4 T-cells observedin the complete HLACs may reflect residual anti-CD4 beads bound toinfected CD4 T-cells.

Example 5 HIV 201-Mediated Fusion is Necessary for Depletion ofNon-Productively Infected CD4 T-Cells

Studies with AMD3100 and AZT indicated that indirect CD4 T-cell killingis mediated by events occurring between gp120-CXCR4 binding and reversetranscription. Engagement of the chemokine coreceptor inducesconformational changes in gp41, resulting in insertion of viral fusionpeptide on gp41 into the target T-cell membrane. To determine if thegp120-CXCR4 interaction alone or later events involving viral fusion arerequired for indirect killing, the effects of enfuvirtide (T20), afusion inhibitor that blocks six-helix bundle formation by gp41, aprerequisite for virion fusion and core insertion, were evaluated.

The optimal concentrations of T20 that block viral infection was firstdetermined (FIG. 4A). In NL4-3-infected cells, T20 began to inhibitinfection at concentrations >2 μg/ml; complete inhibition required 10μg/ml. In cells infected with a primary viral isolate, WEAU 16-8 (FIG.5, below), infection was completely inhibited by 0.1 μg/ml of T20. T20did not inhibit infection with a T20-resistant mutant, SIM (Rimsky etal., 1998, J Virol 72:986-993), regardless of concentration.

Next, it was investigated the effect of T20 on indirect CD4 T-cellkilling (FIG. 4B). In the absence of T20, high levels of indirectkilling were observed. T20 concentrations that blocked infection alsogreatly inhibited indirect killing. T20 did not inhibit indirect killingin cultures containing SIM-infected cells. Thus, blocking gp41-mediatedfusion prevents indirect killing.

Next a T20-dependent mutant, GIA-SKY (Baldwin et al., 2004, J Virol78:12428-12437), which fuses only when T20 is present, but cannotinitiate a spreading infection in the absence of T20 was examined (FIG.4C). Consistent with its T20 dependency, in the presence of 1 μg/ml T20,the GIA-SKY mutant readily replicated while growth was inhibited athigher or lower T20 concentrations. The single-domain mutants GIA andSKY exhibited a T20-resistance phenotype similar to that of SIM.

GIA-SKY-infected cells did not induce indirect killing of CD4 T-cells inthe absence of T20 (FIG. 4D). Indirect killing was observed in culturestreated with 1 μg/ml T20 but was inhibited at higher or lowerconcentrations. Since T20-dependent viruses were bound to CXCR4 beforeT20 was added, these findings argue that CXCR4 signaling is notsufficient to elicit indirect CD4 T-cell killing.

Example 6 High Levels of Indirect Killing by HIV-1 Encoding PrimaryEnvelope from a Rapid AIDS Progressor

To better replicate the conditions leading to the loss ofnon-productively infected CD4 T-cells by HIV-1 in vivo, the indirectkilling potential of primary viral isolates obtained from a patient(WEAU) (Clark et al., 1991, N Engl J Med 324:954-960), who wasclassified as a rapid progressor with high viral loads and a rapiddecline in CD4 T-cell count, was evaluated (FIG. 5A). WEAU 16-8 virionscontained an envelope isolated 16 days after the onset of symptoms thatexhibited dual co-receptor tropism (R5/X4). Uninfected CFSE-labeledcells were co-cultured with cells infected with WEAU 16-8 or laboratoryadapted NL4-3 or with uninfected cells in the presence of AZT alone orwith AMD3100. After 5 days, viable CFSE-positive CD4 and CD8 T-cells andB cells were counted (FIG. 5B). In the presence of AZT alone, depletionof CSFE-positive CD4 T-cells was approximately 20% greater inco-cultures with WEAU 16-8-infected cells than in those withNL4-3-infected cells. In both cases, the number of CFSE-positive CD8 Tand B cells remained essentially unchanged. Thus, primary viruses, likethe laboratory-adapted NL4-3 virus, induce pronounced indirect killingin lymphoid cultures. Notably, AMD3100 prevented indirect killinginduced by the dual-tropic WEAU 16-8 virus, indicating that indirectkilling in lymphoid histocultures is primarily mediated by the CXCR4pathway. TAK779, an antagonist of the CCR5 receptor, had no effect onthe observed indirect killing of CD4 T-cells (data not shown).

Example 7 Indirect Killing Requires a Close Interaction of Uninfectedand HIV-Infected Cells

Next it was examined whether indirect killing requires close contactwith HIV-infected cells or instead can be fully supported by virionsaccumulating in the supernatants of the infected histocultures. It wasfound that cell-free supernatants from HIV-infected histocultures weremuch less efficient at inducing indirect killing (FIG. 6A). To excludethe possibility that the concentration of virions in the supernatantswas too low, the experiment was repeated using a 20-fold concentratedvirion supernatants (1 μg p24/ml) but still failed to detect indirectCD4 T-cell killing (FIG. 6B). Together, these findings suggest thatclose cell-cell contact is likely required for indirect killing.

To further explore the potential requirement of close cell-cell contactfor indirect killing (Sherer et al., 2007, Nat Cell Biol 9:310-315;Sourisseau et al., 2007, J Virol 81:1000-1012), these assays wererepeated using cells that had been washed daily with fresh RPMI toprevent accumulation of HIV-1 virions and soluble factors. Such cellwashing did not affect the ability of the resultant infected cells tomediate indirect CD4 T-cell killing (FIG. 6B), suggesting that virionsreleased into the medium do not participate in indirect killing. Thesefindings were confirmed using a transwell culture system. CSFE-labeledcells and HIV-infected cells were mixed or physically separated by atranswell insert with 1 μm pores, which allows free diffusion of virionsbut not cells. Indirect killing was substantial in the mixed culturesbut not in the transwell cultures (FIG. 6C). Together, these findingsindicated that indirect killing requires close interaction betweenCFSE-labeled and HIV-1-infected cells, consistent with in vitro (Garg etal., 2007, J Biol Chem 282:16899-16906; Holm and Gabuzda, 2005, J Virol79:6299-6311) and in vivo studies showing that apoptoticnon-productively infected cells in human lymph nodes often cluster nearproductively infected cells (Finkel et al., 1995, Nat Med 1:129-134).

Example 8 Indirect Killing Requires Fusion of Virions from NearbyHIV-Producing Cells

Indirect killing required gp41-mediated fusion and close interactionwith HIV-infected cells, suggesting that cell death may be caused by thefusion of HIV-1 virions to CD4 T-cells, syncytia formation, orhemifusion (mixing of lipids in the absence of fusion pore formation)mediated by Env present on HIV-infected cells interacting withneighboring CD4 T-cells. HIV-1 virions (Holm et al., 2004, J Virol78:4541-4551; Jekle et al., 2003, J Virol 77:5846-5854; Vlahakis et al.,2001, J Clin Invest 107:207-215), cell-mediated fusion (LaBonte et al.,2000, J Virol 74:10690-10698; Margolis et al., 1995, AIDS Res HumRetroviruses 11:697-704), and hemifusion (Garg et al., 2007, J Biol Chem282:16899-16906) have been proposed to be involved in indirect killing.Therefore, the requirement for cell-cell interaction in indirect killingmay be mediated either by effective delivery of HIV-1 virions or bycell-associated Env.

To discriminate between virion-mediated and cell-associated Envinduction of indirect killing, the effects of HIV protease inhibitorswere tested. These inhibitors act during the budding process, resultingin immature viral particles that cannot fuse with target cells (Wyma etal., 2004, J Virol 78:3429-3435). The effect of protease inhibitors onviral maturation was assessed first. NL4-3 viruses carrying aβ-lactamase-Vpr (BlaM-Vpr) reporter protein were produced in 293T-cellsin the presence or absence of the HIV protease inhibitor amprenavir. Amutant virus, TR712, encoding a form of gp41 lacking 144 of the 150amino acids in the C-terminal cytoplasmic tail was also produced. Thisdeletion largely relieves the impaired fusogenic properties of immatureHIV-1 particles (Wyma et al., 2004, J Virol 78:3429-3435). Proteinanalysis of viral lysates showed that the NL4-3 and TR712 virionsappropriately cleaved gp160 to generate gp120 in the presence andabsence of amprenavir. However, in the presence of amprenavir, anuncleaved form of p55 Gag polypeptide rather than the mature p24 CAprotein accumulated in both NL4-3 and TR712 virions (FIG. 6D). Theseresults confirm that amprenavir treatment of virus producing cellsresults in the accumulation of immature particles containing normallevels of incorporated Env proteins.

To test the ability of these viruses to fuse with target cells, an HIVvirion-based fusion assay was used that measures β-lactamase (BlaM)activity delivered to target cells upon the fusion of virions containingBlaM fused to the Vpr protein (BlaM-Vpr) (Cavrois et al., 2002, NatBiotechnol 20:1151-1154). Immunoblotting for BlaM confirmed that NL4-3and TR712 virions incorporated Blam-Vpr in the presence or absence ofamprenavir (FIG. 6D).

Next, SupT1 cells were infected with mature or amprenavir-treatedimmature NL4-3 or TR712 virions containing BlaM-Vpr. Immature NL4-3viruses displayed a 90% decline in fusogenic properties (FIG. 6E). Incontrast, immature TR712 retained 40% fusion capacity, indicating thatthe impaired fusion is not a result of a defective BlaM enzyme. Thus,immature virions generated in the presence of amprenavir display greatlyreduced ability to fuse with target cells. Importantly, proteaseinhibitors did not affect the function of Env proteins expressed oninfected cells and did not block cell-cell fusion.

Next the effect of protease inhibitors on indirect killing wasinvestigated. Remarkably, three different protease inhibitors inhibitedindirect killing as efficiently as AMD3100 (FIG. 6F). These resultsindicated that HIV-1 virions, not HIV-infected cells, are responsiblefor indirect CD4 T-cell killing. Additionally, recapitulating theefficient viral delivery of close cell-cell interactions byspinoculation of free virions resulted in extensive and selectiveindirect killing of CD4 T-cells while sparing CD8 T-cells and B cells(FIG. 7A-B). Thus, although indirect killing in lymphoid culturesrequires a close interaction between non-productively and productivelyinfected cells, this killing involves virions rather thancell-associated Env.

Example 9 Extensive and Selective Indirect Killing of CD4 T-Cells bySpinoculation

Data obtained suggested that indirect killing requires efficientdelivery of virions by close cell-cell contact. Notably, theparticle-to-infectivity ratio for HIV-1 was quite low (10⁻³ to 10⁻⁴). Incontrast, the infectivity of virus producing cells, as measured inco-cultured systems, was approximately 10² to 10³ times higher (Dimitrovet al., 1993; J Virol 67:2182-2190). Although the mechanism responsiblefor such distinctive infection capacities is unclear, it is possiblethat TREX1, a cellular 3′ DNA exonuclease plays a role by degradingcytoplasmic reverse transcribed DNA products (Stetson et al., 2008, Cell134:587-598). TREX1 activity in the cytoplasm may create a threshold ofDNA products that must be achieved for the initiation of productiveinfection in permissive cells, or alternatively, to induce cell death inabortively infected cells.

Therefore it was assessed whether the synchronized delivery of largenumbers of HIV-1 particles by spinoculation (FIG. 7A) would generatesufficient incomplete reverse transcripts to induce a cytopathicresponse in CD4 T-cells. Remarkably, spinoculation of HLAC with HIV-1induced extensive and selective depletion of CD4 T-cells (FIG. 7B). Lossof CD4 T-cells was prevented by efavirenz but not by AZT, indicatingthat cell death was due to abortive HIV-1 infection. These datademonstrated that efficient viral delivery was key to recapitulate theextensive indirect killing of CD4 T-cells mediated by close cell-cellinteractions. Additionally, delivering the virions in such synchronousmanner allowed for the evaluation of abortive infection and subsequentcell death in real time. Of note, when high doses of HIV-1 (≧2 μgp24gag/200 μl) are spinoculated, CD4 T-cell killing occurs within 12hours and is not prevented by efavirenz (data not shown), suggestingthat the efavirenz block can be overwhelmed under certain conditions.Nevertheless, T20 prevented CD4 T-cell killing even at such high viraldoses, indicating that the observed indirect cell death involvedabortive infection.

Example 10 Non-Permissive CD4 T-Cells Die from Abortive Infection

Based on these findings, it was hypothesized that “indirect killing”involves an abortive form of infection, like that which occurs innonpermissive resting CD4 T-cells. These naive CD4 T-cells exhibit anearly post-entry block to HIV-1 infection that can be relieved byactivation with phytohemagglutinin (PHA) and interleukin-2 (IL-2)(Kreisberg et al., 2006, J Exp Med 203:865-870; Santoni de Sio andTrono, 2009, PLos One 4:e6571; Unutmaz et al., 1999, J Exp Med189:1735-1746; Zack et al., 1990, Cell 61:213-222). To test thishypothesis, the killing of activated and non-activated CFSE-labeledcells in HLACs was compared.

CFSE-labeled cells were activated with PHA and IL-2 two days beforemixing with effector cells, and contained a large percentage of dividingCD25 and CD69 positive cells. Non-activated (resting) CFSE-labeled cellsdid not divide and typically contained a small percentage of cellsexpressing CD25 and CD69 (FIG. 8A). Either in the presence or absence ofAZT, killing of resting CFSE-labeled CD4 T-cells was robust (FIG. 8B,columns 4+5 and 16+17). In sharp contrast, activated CFSE-labeled CD4T-cells were not depleted in the absence of AZT, but were extensivelydepleted in cultures containing AZT (FIG. 8B, columns 10+11 and 22+23).Addition of AMD3100 prevented the AZT-induced killing of activatedCFSE-labeled cells, excluding non-specific toxic effects of AZT in theactivated cells (FIG. 8B, columns 12 and 24).

The ability of AZT to promote indirect killing of activated CD4 T-cellssuggested that cell death is triggered by impaired reversetranscription. To investigate this possibility, the experiment wasrepeated with two pairs of AZT-resistant HIV-1 clones, 629 and 964(Larder et al., 1989, Science 243:1731-1734). It was first determinedthat concentrations of 0.5 μM AZT block viral replication inNL4-3-infected and AZT-sensitive clones and achieve half maximalinhibitory effect in AZT-resistant clones (FIGS. 9A-B).

When resting CFSE-labeled cells were used, the extent of killing by theAZT-resistant HIV-1 viruses was similar to that obtained with NL4-3 withor without AZT (FIG. 8C resting CFSE-positive cells), demonstrating aredundant function for endogenous termination of reverse transcriptionand AZT. Alternatively, when activated CFSE-labeled cells were tested,AZT-resistant HIV-1 clones did not deplete CFSE-labeled CD4 T-cells inthe presence of AZT (FIG. 8C, columns 29 and 35).

Example 11 Indirect Killing Requires Elongation of Viral DNA inAbortively Infected CD4 T-Cell

In the absence of Vif, human APOBEC3G from virus-producing cells ispackaged into HIV-1 particles. Incorporated APOBEC3G blocks HIV-1infection after initiation of reverse transcription but before thecompletion of strong-stop DNA synthesis (Bishop et al., 2008, PLoSPathog 4, e1000231; Li et al., 2007, J Biol Chem 282:32065-32074),regardless of target cell permissively. Therefore the effect ofvif-deficient (Δvif) HIV-1 on resting and activated CD4 T-cells wasassessed. Because these particles introduce short reverse transcriptionproducts into the target cell, it was tested whether this would besufficient to elicit indirect killing of activated CD4 T-cells even inthe absence of AZT. To test this hypothesis, HLACs were infected withwild type (WT) and Δvif NL4-3 viruses generated in 293T-cells in theabsence of APOBEC3G. Particles in supernatants from HIV-infectedhistocultures were collected after 6 days and subjected to proteinanalysis (FIG. 9C). Equal amounts of WT and Δvif NL4-3 virions wereproduced, as shown by the levels of HIV-1 p24^(gag). However, asexpected, Δvif NL4-3 virions packaged markedly more endogenous APOBEC3Gmolecules than WT virions.

Next, cells infected with WT or Δvif NL4-3 HIV-1 were co-cultured withresting or activated CSFE-labeled cells in the presence or absence ofAZT, NNRTIs, or AMD3100 (FIG. 9D). Resting CFSE-labeled CD4 T-cells werenot extensively depleted by Δvif NL4-3-infected cells when co-culturedwith AZT or no drugs (FIG. 10B, columns 9 and 10 vs. 17 and 18). Theseresults suggested that early termination of DNA synthesis byvirion-incorporated APOBEC3G prevents the cytopathic response ininfected CD4 T-cells.

Of note, Δvif NL4-3-infected cells also did not deplete activatedCFSE-labeled cells in the absence of drugs (FIG. 8, Panel D, column 41).Moreover, when AZT was added, indirect cell depletion was observed onlyin cultures containing cells infected with WT HIV-1 (FIG. 8, Panel D,column 34 vs. 42). These results indicate that the termination ofreverse transcription before the completion of strong-stop DNA synthesisis not sufficient to generate a cytopathic response.

Example 12 Death of Abortively Infected CD4 T-Cells is Triggered byPremature Termination of Viral DNA Elongation

Next it was determined what stage of reverse transcription triggersabortive infection cell death. AZT inhibits DNA elongation but not earlyDNA synthesis (Arts and Wainberg, 1994, Antimicrob Agents Chemother38:1008-1016). It was examined whether blocking early DNA synthesis withnon-nucleoside reverse transcriptase inhibitors (NNRTIs) would have thesame effect as AZT. Impaired reverse transcription may also lead toabortive integration, causing chromosomal DNA breaks and a genotoxicresponse. To exclude this possibility, integrase inhibitors were used.To discriminate between the cytopathic response induced by endogenoustermination of reverse transcription and the response induced by AZT,resting and activated CFSE-labeled cells were separately assessed.

Remarkably, the NNRTIs, efavirenz and nevirapine, blocked indirectkilling of resting CD4 T-cells as efficiently as AMD3100 (FIG. 8D,columns 15 and 16). These findings suggested that allosteric inhibitionof reverse transcriptase induced by these NNRTI's interrupts reversetranscription sufficiently early to abrogate the death response. Incontrast, the integrase inhibitors raltegravir and 118-D-24 did notprevent abortive infection killing (FIG. 8D, columns 17 and 18),suggesting that cell death involves signals generated prior to viralintegration. NNRTIs also protected activated CFSE-labeled cells fromdeath induced by AZT (FIG. 8D, column 38 vs. columns 44 and 45),demonstrating that a certain degree of DNA synthesis is required toelicit the cytopathic response.

This notion was further strengthened in findings obtained withvif-deficient (Δvif) HIV-1 particles where reverse transcription isinhibited during strong-stop DNA synthesis due to incorporated APOBEC3G(A3G) (Bishop et al., 2008, PLos Pathog 4:e1000231; Li et al., 2007, JBiol Chem 282:32065-32074). Abortively infected CD4 T-cells were notdepleted by Δvif NL4-3-infected cells (FIG. 9C-D), indicating thattermination of reverse transcription before the completion ofstrong-stop DNA synthesis is not sufficient to generate a cytopathicresponse. Other HIV-1 mutants containing substitutions in RNase H andnucleocapsid that promote early defects in reverse transcription failedto elicit indirect CD4 T-cell killing (FIGS. 9E-F). Together, thesefindings indicate that accumulation of reverse-transcribed DNA, ratherthan any inherent activity of the HIV-1 proteins, is the key factor thattriggers the death response.

Example 13 Reverse-Transcribed Viral DNA is Required for Killing ofAbortively Infected CD4 T-Cells

To further confirm the role of viral DNA synthesis in indirect killing,viruses harboring genetic mutations that interrupt reverse transcriptionwere examined. First, the clone E478Q contains a point mutation in thecatalytic site of the RNase H domain of reverse transcriptase thatcompromises its RNase H activity. It fuses into the target cells butcannot reverse transcribe beyond the very early strong-stop products(Smith et al., 1999, J Virol 73:6573-6581). Second, the pDB653 proviralclone contains mutations in the zinc finger domains of the nucleocapsid(NC) protein and is defective in reverse transcription synthesis andviral RNA packaging (Guo et al., 2000, J Virol 74:8980-8988). Becausethese viruses are not competent for multiple rounds of viralreplication, it was not possible to use traditional HLAC cultureconditions. Instead, the system was modified by overlaying HLAC cells ona monolayer of 293T-cells that had been transfected with these proviralclones (FIG. 9E). Using this approach which did not require a spreadingviral infection, extensive and selective depletion of HLAC CD4 T-cellswas observed when 293T-cells were transfected with NL4-3 (FIG. 9F). Lossof CD4 T-cells occurred both in the presence and absence of AZT, but wascompletely blocked by addition of efavirenz or AMD3100 (FIG. 9, panel F,columns 5-8), indicating that the observed cell death involves abortiveHIV-1 infection. Transfection of E478Q and pDB653 HIV-1 clones did notresult in depletion of CD4 T-cells (FIG. 9, Panel F, columns 9-11 and13-16, respectively), indicating that RNA-directed DNA synthesis is keyfor generating a cytopathic response.

Further, the TR712 HIV-1 clone that encodes a truncated c-terminaldomain in gp41 was examined. Transfections with the TR712 HIV-1 clonemarkedly depleted HLAC CD4 T-cells (FIG. 9, panel F, column 17-20).Because immature TR712 viruses retain their capacity to fuse in thepresence of protease inhibitors, the ability of such immature TR712viruses to mediate killing of CD4 T-cells was tested. To this end, theexperiment with the TR712 clone in the presence of the proteaseinhibitor amprenavir was repeated. This process generated fusioncompetent virions containing immature, unprocessed capsid proteins.Remarkably, amprenavir prevented CD4 T-cell killing by TR712 viruses(FIG. 9, panel F, columns 21-24), suggesting that fusion of immatureparticles do not induce a cytopathic response. Previous studies haveshown that HIV particles with irregular core morphologies (Tang et al.,2001, J Virol 75:9357-9366) and altered stabilities (Fitzon et al.,2000, Virology 268:294-307; Stremlau et al., 2004, Nature 427:848-853)are unable to undergo reverse transcription in cells. Hence, reversetranscription of immature TR712 viruses was likely disrupted afterentry, preventing induction of the cytopathic response. Together, theseresults suggested that accumulation of reverse-transcribed DNA, ratherthan the mere delivery of HIV-1 components into CD4 T-cells, is the keyfactor that initiates the cytopathic response.

Example 14 HIV-1 Env-Receptor Interactions are not Required for IndirectKilling of CD4 T-Cells

Some models of indirect CD4 T-cell death have implicated HIVEnv-receptor interactions (Holm et al., 2004, J Virol 78:4541-4551;Jekle et al., 2003, J Virol 77:5846-5854; Perfettini et al., 2005, CellDeath Differ 12 Suppl 1:916-923; Vlahakis et al., 2001, J Clin Invest107:207-215; Vlahakis et al., 2003, J Infect Dis 188: 1455-1460). Instudies exploring whether HIV-1 Env was essential, it was first foundthat transfection of an HIV-1 clone lacking the Env gene (NL4-3 Δenv)failed to deplete HLAC CD4 T-cells (FIG. 9, panel F, columns 25-28).However, replacement of HIV Env with the amphotropic Env of MoloneyMurine Leukemia Virus (MLV) restored CD4 T-cell killing (FIG. 9, panelF, columns 29-30), demonstrating that cell death does not obligatoryrequire HIV-1 Env binding to its surface receptors. In contrast,co-expression of the vesicular stomatitis virus glycoprotein (VSV-G),which mediates viral entry within acidified endosomes, did not result inCD4 T-cell killing, These findings are in agreement with previousstudies showing that VSV-G fuses very poorly to resting CD4 T-cells (Yuet al., 2009, PLoS Pathog 5:e1000633). Thus, although HIV-1 Env-receptorinteractions are not required for indirect killing, these findingshighlighted the importance of the HIV envelope for infection of restinglymphoid CD4 T-cells.

Example 15 Abortively Infected CD4 T-Cells Commence but do not CompleteReverse Transcription

Next the status of HIV-1 reverse transcription in tonsillar CD4 T-cellsafter infection was examined. Specifically, the effect on reversetranscription after treatment with NNRTIs, such as efavirenz andnevirapine, which prevent the death of abortively infected CD4 T-cells,or with AZT or integrase inhibitor (raltegravir) that do not prevent CD4T-cell death, was investigated. Taqman-based quantitative real-time PCR(QPCR) was used to quantify the synthesis of reverse transcriptionproducts in isolated CD4 T-cells from HLAC 16 hours after infection withNL4-3. Specific QPCR primers and probes were designed (Table 1) tomonitor sequential steps in reverse transcription including generationof strong-stop DNA, first template exchange (Nef), and DNA strandelongation (Env) (FIG. 10A)

TABLE 1 The primer and probe sets used for Taqman- based quantitativereal-time PCR of HIV-1 reverse transcription cDNA products Strong-stop(R/U5) region F42 5′-GGCTAACTAGGGAACCCACTGC-3′ SEQ ID NO: 39 R985′-CAACAGACGGGCACACACTACT-3′ SEQ ID NO: 40 P65 5′-(6~FAM)- SEQ ID NO: 41TAAGCCTCAATAAAGCTTGCCTTGAGTGCTC- (MGBNFQ)-3′ Nef (Nef/U3) region. Thisamplicon is located 170 nucleotides downstream to the repeat (R)sequence, after cDNA synthesis proceeds first strand transfer F5545′-TTGACAGCCGCCTAGCATT-3′ SEQ ID NO: 42 F591 5′-TTGAAGTACTCCGGATGCAGC-3′SEQ ID NO: 43 P574 5′-(6~FAM)-CATCACGTGGCCCGAG- SEQ ID NO: 44(MGBNFQ)-3′ Env region. This amplicon is located 3023 nucleotidesdownstream to the repeat (R) sequence, after cDNA synthesis proceedsfirst strand transfer. F286 5′-TGGACAAATGACATGGTAGAACAGA-3′ SEQ ID NO:45 R339 5′-TTTACACATGGCTTTAGGCTTTGA-3′ SEQ ID NO: 46 P3135′-(6~FAM)-CATGAGGATATAATCAG SEQ ID NO: 47 TTTATGG-(MGBNFQ)-3′

Reverse transcription products corresponding to strong-stop DNA weresimilar in untreated CD4 T-cells or cells treated with AZT, NNRTIs, orraltegravir but were greatly reduced in cells treated with AMD3100 or incultures infected with Δvif NL4-3 where arrest occurs prior to thecompletion of strong-stop DNA synthesis (FIG. 10B, columns 1-8). Incontrast, the accumulation of later reverse transcription productsdetected by the Nef and Env probes were dramatically inhibited by theNNRTIs but not by raltegravir. Levels of Nef (FIG. 10B, columns 10+11)and Env (columns 18+19) DNA products were similar in untreated cells andcells treated with AZT, indicating that reverse transcription in mosttonsillar CD4 T-cells naturally terminates during DNA chain elongation,coinciding with the block induced by AZT. The minor inhibition detectedby AZT is likely due to a small number of permissive CD4 T-cells in theculture. These results show that abortively infected CD4 T-cellsaccumulate incomplete reverse transcription products representative ofDNA strand elongation. Blocking earlier steps of reverse transcriptionby NNRTIs or by genetic mutations like deletion of Vif or mutation ofRNase H restricts accumulation of such products, and prevents abortiveinfection-induced cell death (FIG. 11A).

Example 16 DNA Reverse Transcription Intermediates Elicit a CoordinatedProapoptotic and Proinflammatory Response in Abortively Infected CD4T-Cells

Next it was evaluated whether HIV-mediated indirect killing of CD4T-cells is associated with deregulation of cytokine production or a DNAdamage response. To facilitate a vigorous and synchronized killingeffect, HLACs were spinoculated with NL4-3 virions in the presence ofvarious antiviral drugs. Interestingly, based on immunostaining aftercytokine capture, abortively infected CD4 T-cells expressed IFN-α, andhigh levels of the proinflammatory interleukin 1β (IL-1β), but not tumornecrosis factor (TNFα) (FIG. 10C). Phosphorylation of S37 p53 was notobserved, suggesting that abortive HIV-1 infection does not induce a DNAdamage cascade. Abortively infected CD4 T-cells also displayed caspase-1and caspase-3 activity along with appearance of annexin V (FIG. 10D).T20 and efavirenz but not AZT prevented activation of these caspases,indicating that apoptosis was induced by abortive HIV-1 infection. Celldeath was completely prevented by Z-VAD-FMK, a pan-caspase inhibitor,suggesting that caspase activation is required for the observedcytopathic response (FIG. 10E). Such mode of cytokine production andcaspase activation was not observed in CD8 T or B cells.

Next it was examined whether abortive HIV-1 infection signals for thematuration and secretion of IL-1β. In cells IL-1β activity is rigorouslycontrolled. Cells can be primed to express inactive pro-IL-1β by variousproinflammatory signals. However, the release of bioactive IL-1βrequires a second signal leading to activation of inflammasomes,cleavage of pro-IL-1β by caspase-1 and secretion of the bioactive 17 kDaform of IL-1β (Schroder and Tschopp, 2010, Cell, 140:821-832).Interestingly, western blot analysis revealed high amounts ofintracellular pro-IL-1β in untreated CD4 T-cells, suggesting thattonsillar CD4 T-cells are primed to release proinflammatory mediators(FIG. 10F). Stimulating the CD4 T-cells with PMA and nigericin inducedfurther accumulation of pro-IL-1β and promoted the maturation andrelease of the bioactive 17 kDa IL-1β into the supernatant. Remarkably,infection of CD4 T-cells with NL4-3 in the presence of AZT similarlyresulted in maturation and release of the bioactive 17 kDa IL-1β intothe supernatant. This response was completely prevented by efavirenz andAMD3100, suggesting that abortive HIV-1 infection signals the maturationand release of bioactive IL-1β in these CD4 T-cells.

To identify the nature of the nucleic acid species that trigger theseresponses, a recently described H35 rat hepatocyte cell line containingan IFN-sensitive response element (ISRE) linked to GFP (Patel et al.,2009, Proc Natl Acad Sci USA 106:12867-12872) was used. H35 cells werefirst infected with pseudotyped VSV-G HIV-1 virions. These virionsinduced GFP expression and cell death in the presence or absence of AZT.Importantly, the expression of both GFP and cell death response wereblocked by efavirenz but not raltegravir (FIG. 13D). Thus, the H35system successfully reconstitutes the cytokine and cytopathic responseobserved in tonsillar CD4 T-cells. Next the various HIV-1 reversetranscription intermediates were synthesized and tested for theirability to activate the ISRE-GFP reporter. Interestingly, none of theRNA-containing oligonucleotides stimulated the ISRE-GFP reporterexpression above baseline. In sharp contrast, ssDNA and dsDNAoligonucleotides longer than 500 bases in length, which corresponded toreverse transcription intermediates produced during DNA elongation,evoked a potent ISRE-GFP activation (FIG. 10G). Similarly, when cellswere stimulated with poly(I:C), a synthetic double-stranded RNA known toactivate IRF3 via the RIG-I pathway elicited a comparable ISRE-GFPresponse. Taken together, these findings indicate that reversetranscription intermediates generated during DNA chain elongation inducea coordinated proapoptotic and proinflamatory innate immune responseinvolving caspase-3 and caspase-1 activation in abortively infected CD4T-cells.

Example 17 Abortive HIV-1 Infection Represents a General Mechanism ofCD4 T-Cell Depletion in Human Lymphoid Organs

To confirm that the cytopathic effect mediated by abortive HIV-1infection is not limited to tonsillar tissue, indirect CD4 T-cellkilling in HLACs formed from fresh human splenic tissue was assessed. Ofnote, effector spleen cells were completely refractory to HIV infection(data not shown), which necessitated activation with PHA and IL-2 beforeinfection with NL4-3. Nevertheless, non-activated CSFE-labeled spleenCD4 T-cells were extensively depleted in cultures containingHIV-infected spleen HLACs (FIG. 11 B, C). Loss of CFSE-labeled CD4T-cells was robust in the absence of drugs or in the presence of AZT,indicating that productive viral replication was not required for CD4T-cell death in spleen. Remarkably, addition of efavirenz prevented theloss of CD4 T-cells as efficiently as AMD3100, indicating that celldeath involved abortive infection. The integrase inhibitors raltegravirdid not prevent CD4 T-cell killing demonstrating that signaling for celldeath occurs before viral integration.

Lymphocytes continuously circulate between one peripheral lymphoid organand another via the lymph and blood. After leaving one organ andentering second, productively infected CD4 T-cells may come into contactwith a new pool of uninfected lymphocytes. To simulate these conditions,infected human tonsil cells were co-cultured with resting(non-activated) CFSE-labeled human spleen cells. It was observed thatresting spleen CD4 T-cells were massively depleted by HIV-infectedtonsil cells in the absence of drugs or in the presence of AZT. Further,it was found that the addition of efavirenz, but not raltegravir,prevented this loss of CD4 T-cells. These findings support a mechanismof cell death involving abortive HIV infection. Taken together, theseresults demonstrated that abortive HIV infection is a general mechanismof CD4 T-cell depletion, which plays a significant role in the overallcytopathicity induced by HIV-1.

Example 18 HIV-1 Infection Activates Innate Proapoptotic andProinflammatory Responses in Human Lymphoid CD4 T-Cells

Human lymphoid aggregate cultures (HLACs, Doitsh et al., 2010, Cell,143(5):789-801) were left uninfected or infected with NL4-3, anX4-tropic strain of HIV-1, in the presence of AZT (5 μM), efavirenz (100nM), or T20 (10 μg/ml), as indicated. After 3 days, infected CD4 T-cellsdisplayed evidence of caspase-1 and caspase-3 activity and annexin Vpositivity, but caspase 6, 8, and 9 were not activated. Of note, T20 andefavirenz but not AZT prevented activation of these caspases, indicatingthat apoptosis was induced by non-productive HIV-1 infection. Data forthis experiment are depicted in FIGS. 13A and 14B, C.

Example 19 Death of HIV-Infected CD4 T-Cells Requires Caspase Activation

Different members of the caspase family play key roles in inflammationand mammalian apoptosis. The proapoptotic caspases are comprised ofcaspase-3, -6 and -7, while the proinflammatory caspases correspond tocaspase-1, -4 and -5. Caspase inhibitors were used to assess whethercaspase activation is required for indirect CD4 T-cell killing.HIV-infected HLACs were cultured in the presence of Z-VAD-FMK (apan-caspase inhibitor) or Z-FA-FMK (negative control). CD4 T-cell deathwas blocked by Z-VAD-FMK but persisted in the presence of Z-FA-FM.Values are mean±SEM of three experiments with cells from three HLACdonors. Remarkably, CD4 T-cell death was completely prevented byaddition of Z-VAD-FMK, a general caspase inhibitor (FIGS. 13B, 14A).This finding indicated that activation of one or more caspase wasrequired to induce cell death. Interestingly, specific inhibition ofcaspase-3 with Z-DEVD-FMK reduced cell death by only 50% raising thepossibility that multiple caspase signaling cascades may be involved inthe observed killing of CD4 T-cells. These studies further revealed thatcaspase-1 is activated in response to abortive HIV-1 infection, raisinga possible role for both caspase-3 and caspase-1 in CD4 T-cellscytopathology.

Inflammasome-dependent caspase-1 activity can result in a highlyinflammatory form of cell death known as pyroptosis, which results incleavage of IL-1β and IL-18 and early changes in membrane permeabilityleading to release of these inflammatory cytokines. Pyroptosis occursmost frequently upon infection with intracellular pathogens (Schroderand Tschopp, 2010, Cell, 140:821-832)

Example 20 HIV-1 Infection Promotes the Maturation and Secretion ofIL-1β in Lymphoid CD4 T-Cells

In panel FIG. 13C, it is shown that infected CD4 T-cells also displayedIFN-β expression and high-level expression of IL-1β but not TNFα. Theabsence of phospho-S37 p53 suggested that abortive HIV-1 infection doesnot induce a DNA damage cascade. In panel FIG. 13D, it is shown thatisolated tonsillar CD4 T-cells were left untreated or stimulated with0.5 μM PMA (to further induce intracellular stores of 35-kDa pro-IL-1β)and 10 μM nigericin, (a potassium ionophore used as a secondinflammatory stimulus to trigger maturation and release of the bioactive17-kDa IL-1β in primed cells). Cultures were infected with NL4-3 in thepresence of the indicated antiviral drugs. After 3 days, half the cellswere lysed and analyzed by SDS-PAGE and immunoblotting with anti IL-1βand anti-β-actin (loading control). On day 5, supernatants from theremaining cells were analyzed by SDS-PAGE and immunoblotting withanti-IL-1β antibodies. Untreated CD4 T-cells displayed high levels ofintracellular pro-IL-1β consistent with their primed status. Infectionwith NL4-3 in the presence of AZT also resulted in release of bioactive17-kDa IL-1β, as observed after treatment with PMA+nigericin. Thisresponse was blocked by efavirenz and AMD3100 (250 nM). Thus,non-productive HIV-1 infection induces maturation and release ofbioactive IL-1β in lymphoid CD4 T-cells. Data for this experiment aredepicted in FIGS. 13C and 13D.

Example 21 Pro-IL-1β is Abundantly Expressed in Lymphoid CD4 T-Cells butnot in CD8 T or B Cells

Levels of intracellular pro-IL-1β were assessed in HLACs from freshtonsils and spleen tissue from different donors (FIG. 13E). Asterisksindicate samples in which dead cells were removed by Ficoll-Hypaquegradient centrifugation. CD4 T, CD8 T, and B cells were isolated fromdonor 2100 by positive selection with microbeads and analyzed forpro-IL-1β expression. High levels of intracellular pro-IL-1β were foundin purified CD4 T-cells but not in CD8 T or B cells (rectangle). Datafor this experiment are depicted in FIG. 13E.

Example 22 Caspase-1 Inhibitors Efficiently Inhibit Inflammation inHuman Lymphoid CD4 T-Cells

Isolated tonsillar CD4 T-cells were treated overnight with nigericin (10μM) to provide a second inflammatory stimulus, resulting in maturationand release of bioactive 17-kDa IL-1β. Supernatants from the cellcultures were subjected to SDS-PAGE and immunoblotting. 17-kDa IL-1β wasreleased after treatment with nigericin, but not with calcium ionophoreA23187 or the nonspecific cation ionophore monensin. Thus, while thesecells are primed, they must receive the appropriate proinflammatorysignal for pro-IL-1β processing and release. IL-1β release was blockedby pre-treatment with the pan-caspase inhibitor Z-VAD and by thecaspase-1 inhibitors Z-WEHD (SEQ ID NO: 12) and Z-YVAD (SEQ ID NO: 15),consistent with inflammasome-associated caspase-1 processing of IL-1β.Data for this experiment are depicted in FIG. 13F.

Example 23 Caspase-1 Inhibitors Efficiently Inhibit CD4 T-Cell Death inHIV-1-Infected Human Lymphoid Tissues

HLACs were left uninfected or infected with HIV-1 (NL4-3 clone) in thepresence of AZT, efavirenz, AMD3100, or caspase inhibitors (20 μM each),as indicated in FIG. 16. After 3 days, the percentages of viable CD4 Tcells in the cultures were determined by flow cytometry. CD4 T-celldeath in HIV-1-infected cultures was prevented by the pan-caspaseinhibitor Z-VAD (“Pan-Caspase” in FIG. 16A) and by the caspase-1inhibitor (Z-WEHD, “Caspase 1” in FIG. 16A) as efficiently as byefavirenz and AMD3100, but not by the Z-FA-FM (commercial negativecontrol; “Control” in FIG. 16A) and caspase-6 inhibitor. Treatment withcaspase-3 inhibitor (Z-DEVD; “Caspase 3” in FIG. 16A) prevented thedeath of only 50% CD4 T-cell population.

HLACs were left uninfected or infected with NL4-3 in the presence of nodrugs, efavirenz, AMD3100, or caspase inhibitors (50 μM or 100 μM), asindicated in FIG. 16B. After 3 days, the percentages of viable CD4 Tcells in the cultures were determined by flow cytometry. CD4 T-celldeath in HIV-infected cultures was prevented by the caspase-1 inhibitor(Caspase-II inhibitor, Calbiochem; “Caspase 1” in FIG. 16B) as efficientas by efavirenz and AMD3100. In these experiments, treatment with thecaspase-3 inhibitor (Z-DEVD; “Caspase” 3 in FIG. 16B) did not preventthe death of HIV-1-infected CD4 T cells.

Caspase-1 activation can lead to a highly inflammatory form of celldeath called pyroptosis. To gain insight into how CD4 T cells die, itwas determined whether HIV infection in CD4 T cells induce pyroptosiscell death, as measured by lactate dehydrogenase (LDH) release. HLACswere left uninfected or infected with NL4-3 in the presence of no drugs,efavirenz, AMD3100, or caspase inhibitors (50 μM or 100 μM), asindicated in FIG. 16C. After 3 days, the supernatants were assayed forthe cytosolic enzyme lactate dehydrogenase (LDH) and the LDH released bydying cells was quantified. HIV-1-infected CD4 T cells released highlevels of LDH, compared to uninfected cells FIG. 16C). These datasuggest that pyroptosis is the predominant form of CD4 T-cell deathafter HIV-1 infection. LDH was not released from infected cells treatedwith efavirenz. AMD3100, or caspase-1 inhibitor (Caspase-II inhibitor,Calbiochem; “Caspase 1 in FIG. 16C). Treatment with caspase-3 inhibitor(Z-DEVD; “Caspase 3” in FIG. 16C) did not prevent the LDH release fromHIV-1 infected cells.

Example 24 Perspective

The mechanism through which HIV-1 kills CD4 T-cells, a hallmark of AIDS,has been a topic of vigorous research and one of the most pressingquestions for the field over the last 28 years (Thomas, 2009; Nat Med15:855-859). In this study, Applicants investigated the mechanism ofHIV-1-mediated killing in lymphoid tissues, which carry the highestviral burdens in infected patients. Applicants used HLACs formed withfresh human tonsil cells and an experimental strategy that clearlydistinguishes between direct and indirect mechanisms of CD4 T-celldepletion. Applicants now demonstrate that indirect cell killinginvolving abortive HIV infection of CD4 T-cells accounts for the vastmajority of cell death occurring in lymphoid tissues. No more than about5% of the CD4 T-cells are productively infected, but virtually all theremaining CD4 T-cells are abortively infected ultimately leading tocaspase-mediated cell death. Equivalent findings were observed in HLACsformed with fresh human spleen (FIGS. 11B-C), indicating this mechanismof CD4 T-cell depletion can be generalized to other lymphoid tissues.

The massive depletion of non-productively infected CD4 T-cells is incontrast to their survival after infection of intact blocks of tonsillartissue in human lymphoid histoculture (HLH) (Drivel et al., 2003; AIDSRes Hum Retroviruses 19:211-216). This result probably reflectsdifferences between the HLH and the HLAC experimental systems. In HLH,the complex three-dimensional spatial cellular organization of lymphoidtissue is preserved, but cellular movement and interaction arerestricted, both of which are required for indirect killing. In HLAC,the tissue is dispersed, and cells are free to interact, resulting in arapid and robust viral spread. While the mechanism triggering indirectCD4 T-cell death is certainly identical in both settings, HLH allowsonly a slow, nearly undetectable progression of indirect CD4 T-celldeath. In HLAC, this process is accelerated, allowing the outcome to bedetected in a few days. Interestingly, indirect killing was also lessefficient when peripheral blood cells were tested (data not shown). Itis possible that cellular factors specifically produced in lymphoidorgans are required to accelerate indirect killing of peripheral bloodCD4 T-cells.

Several mechanisms have been proposed to explain indirect CD4 T-cellkilling during HIV infection. Applicants' finding that CD4 T-cell deathis blocked by entry and fusion inhibitors but not by AZT, stronglysuggested that such killing involves non-productive infection of CD4T-cells. Therefore, Applicants focused on events that occur after HIV-1entry. Our investigations demonstrate that abortive viral DNA synthesisoccurring in nonpermissive, quiescent CD4 tonsil T-cells, plays a keyrole in the cell death response. Conversely, in the small subset ofpermissive target cells, reverse transcription is not interrupted,minimizing the accumulation and subsequent detection of such reversetranscription intermediates (FIG. 7).

Interrupted or slowed reverse transcription may create persistentexposure to cytoplasmic DNA products that elicit an antiviral innateimmune response coordinated by activation of type I IFNs (Stetson andMedzhitov, 2006; Immunity 24:93-103). Such activation, termedIFN-stimulatory DNA (ISD) response, may be analogous to the type I IFNresponse triggered by the RIG-I-like receptor (RLR) family of RNAhelicases that mediate a cell-intrinsic antiviral defense (Rehwinkel andReis e Sousa, 2010; Science 327:284-286). The results herein suggestthat abortive HIV-1 infection also stimulates activation of caspase-3,which is linked to apoptosis, and caspase-1, which promotes theprocessing and secretion of the proinflammatory cytokines like IL-1β. Itis certainly possible that pyroptosis elicited in response to caspase-1activation also contributes to the observed cytopathic response(Schroder and Tschopp, 2010; Cell 140:821-832). The release ofinflammatory cytokines during CD4 T-cell death could also contribute tothe state of chronic inflammation that characterizes HIV infection. Thisinflammation may fuel further viral spread by recruiting uninfectedlymphocytes to the inflamed zone. While this innate response was likelydesigned to protect the host, it is subverted in the case of HIVinfection and importantly contributes to the immunopathogenic effectscharacteristic of HIV infection and AIDS.

Such antiviral pathways comprise an unrecognized cell-intrinsicretroviral detection system (Manel et al., 2010, Nature 467:214-217;Stetson et al., 2008, Cell 134:587-598). Viral RNA in infected cells isrecognized by members of the RIG-I-like family of receptors that detectspecific RNA patterns like uncapped 5′ triphosphate (Rehwinkel and Reise Sousa, 2010, Science 327:284-286). Although uncapped RNA intermediatesare generated by the HIV-1 RNase H, they contain a 5′ monophosphate andtherefore may be not recognized by the RIG-I system (FIG. 10G). Incontrast to RNA receptors, intracellular sensing of viral DNA remainspoorly understood. Consequently, it is unclear how HIV-1 DNAintermediates are detected in the cytoplasm of abortively infected CD4T-cells. AIM2 (absent in melanoma 2) was recently identified as acytoplasmic dsDNA receptor that induces cell death in macrophagesthrough activation of caspase-1 in inflammasomes (Hornung et al., 2009,Nature 458:514-518). Applicants' preliminary investigations have notsupported a role for AIM2 in cell death induced by abortive HIVinfection (not shown) suggesting the potential involvement of adifferent DNA-sensing mechanism.

In summary, both productive and nonproductive forms of HIV infectioncontribute to the pathogenic effects of this lentivirus. The relativeimportance of these different cell death pathways might well vary withthe stage of HIV infection. For example, direct infection and deathmight predominate during acute infection where CCR5-expressing memoryCD4 T-cells in gut-associated lymphoid tissue are effectively depleted.Conversely, the CXCR4-dependent indirect killing we describe in tonsiltissue may reflect later stages of HIV-induced disease where a switch toCXCR4 coreceptor usage occurs in approximately 50% of infected subjects.The current study demonstrates how a cytopathic response involvingabortive viral infection of resting nonpermissive CD4 T-cells can leadnot only to CD4 T-cell depletion but also to the release ofproinflammatory cytokines. The ensuing recruitment of new target cellsto the site of inflammation may fuel a vicious cycle of continuinginfection and CD4 T-cell death centrally contributing to HIVpathogenesis.

All publications, including but not limited to patents and patentapplications, cited in this specification and in the specifications ofthe corresponding priority applications, U.S. Appl. Ser. No. 61/572,883,filed Jul. 22, 2011, U.S. Appl. Ser. No. 61/511,023, filed Jul. 23,2011, and U.S. Appl. Ser. No. 61/575,324, filed Aug. 17, 2011, to theextent that they provide exemplary procedural or other detailssupplementary to those set forth herein, are specifically incorporatedherein by reference as if each individual publication were specificallyand individually indicated to be incorporated by reference herein asthough fully set forth.

1.-20. (canceled)
 21. A method for ameliorating a symptom of HIV-1infection in a patient having an HIV-1 infection, the method comprisingthe step of: (a) administering to a patient having an HIV-1 infection apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound having the formula:

wherein Y is

R¹ is H, C₁₋₁₂aliphatic, C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl,(C₃₋₁₀cycloalkyl)-(C₁₋₁₂aliphatic)-, cycloalkenyl-(C₁₋₁₂aliphatic)-,(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or (5-10 memberedheteroaryl)-(C₁₋₁₂aliphatic)-, wherein any hydrogen atom is optionallyand independently replaced by R⁸ and any set of two hydrogen atoms boundto the same atom is optionally and independently replaced by carbonyl;Ring A is

wherein, in each ring, any hydrogen atom is optionally and independentlyreplaced by R⁴ and any set of two hydrogen atoms bound to the same atomis optionally and independently replaced by carbonyl; when Ring A is

then R is R³C(O)—, HC(O), R³SO₂—, R³OC(O), (R³)₂NC(O), (R³)(H)NC(O),R³C(O)C(O)—, R³—, (R³)₂NC(O)C(O), (R³)(H)NC(O)C(O), or R³OC(O)C(O)—; andR³ is C₁₋₁₂aliphatic, C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl,(C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic)-, (C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-,(5-10 membered heterocyclyl)-(C₁₋₁₂aliphatic)-, or (5-10 memberedheteroaryl)-(C₁₋₁₂aliphatic)-; or two R³ groups bound to the same atomform together with that atom a 3-10 membered aromatic or nonaromaticring; wherein any ring is optionally fused to an C₆₋₁₀aryl, 5-10membered heteroaryl, C₃₋₁₀cycloalkyl, or 5-10 membered heterocyclyl;wherein up to 3 aliphatic carbon atoms may be replaced by a groupselected from O, N, NR⁹, S, SO, and SO₂, wherein R³ is substituted withup to 6 substituents independently selected from R^(8′); when Ring A is

then R is R³C(O)—, as shown below:

and R³ is phenyl, thiophene, or pyridine, wherein each ring isoptionally substituted with up to 5 groups independently selected fromR^(8′), and wherein at least one position on the phenyl, thiophene, orpyridine adjacent to bond x is substituted by R¹², wherein R¹² has nomore than 5 straight-chained atoms; R⁴ is halogen, —OR⁹, —NO₂—CN—CF₃,—OCF₁, —R⁹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁹)₂, —SR⁹, —SOR⁹,—SO₂R⁹—SO₂N(R⁹)₂, —SO₃R⁹, —C(O)R⁹, —C(O)C(O)R⁹, —C(O)C(O)OR⁹,—C(O)C(O)N(R⁹)₂, —C(O)CH₂C(O)R⁹, —C(S)R⁹, —C(S)OR⁹, —C(O)OR⁹, —OC(O)R⁹,—C(O)N(R⁹)₂, —OC(O)N(R⁹)₂, —C(S)N(R⁹)₂, —(CH₂)₀₋₂ NHC(O)R⁹,—N(R⁹)N(R⁹)COR⁹, —N(R⁹)N(R⁹)C(O)OR⁹, —N(R⁹)N(R⁹)CON(R⁹)₂, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂N(R⁹)₂, —N(R⁹)C(O)OR⁹, —N(R⁹)C(O)R⁹, —N(R⁹)C(S)R⁹,—N(R⁹)C(O)N(R⁹)₂, —N(R⁹)C(S)N(R⁹)₂—N(COR⁹)COR⁹, —N(OR⁹)R⁹,—C(═NH)N(R⁹)₂, —C(O)N(OR⁹)R⁹, —C(═NOR⁹)R⁹, —OP(O)(OR⁹)₂, —P(O)(R⁹)₂,—P(O)(OR⁹)₂, or —P(O)(H)(OR⁹); R² is —C(R⁵)(R⁶)(R⁷), C₆₋₁₀ aryl, 5-10membered heteroaryl, or C₃₋₇ cycloalkyl; R⁵ is H or a C₁₋₆straight-chained or branched alkyl; R⁶ is H or a C₁₋₆ straight-chainedor branched alkyl; R⁷ is —CF₃, —C₃₋₇cycloalkyl, C₆₋₁₀aryl, 5-10 memberedheteroaryl, heterocycle, or a C₁₋₆ straight-chained or branched alkyl,wherein each carbon atom of the alkyl is optionally and independentlysubstituted with R¹⁰; or R⁵ and R⁷ taken together with the carbon atomto which they are attached form a 3-10 membered cycloaliphatic; R⁸ andR^(8′) are each independently halogen, —OR⁹, —NO₂, —CN, —CF₃, —OCF₃,—R⁹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁹)₂, —SR⁹, —SOR⁹,—SO₂R⁹, —SO₂N(R⁹)₂—SO₃R⁹, —C(O)R⁹, —C(O)C(O)R⁹, —C(O)C(O)OR⁹,—C(O)C(O)N(R⁹)₂, —C(O)CH₂C(O)R⁹, —C(S)R⁹, —C(S)OR⁹, —C(O)OR⁹, —OC(O)R⁹,—C(O)N(R⁹)₂, —OC(O)N(R⁹)₂, —C(S)N(R⁹)₂, —(CH₂)₀₋₂NHC(O)R⁹,—N(R⁹)N(R⁹)COR⁹, —N(R⁹)N(R⁹)C(O)OR⁹, —N(R⁹)N(R⁹)CON(R⁹)₂, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂N(R⁹)₂, —N(R⁹)C(O)OR⁹, —N(R⁹)C(O)R⁹, —N(R⁹)C(S)R⁹,—N(R⁹)C(O)N(R⁹)₂, —N(R⁹)C(S)N(R⁹)₂, —N(COR⁹)COR⁹, —N(OR⁹)R⁹,—C(═NH)N(R⁹)₂, —C(O)N(OR⁹)R⁹, —C(═NOR⁹)R⁹, —OP(O)(OR⁹)₂, —P(O)(R⁹)₂,—P(O)(OR⁹)₂, and —P(O)(H)(OR⁹); R⁹ is hydrogen, C₁₋₁₂aliphatic,C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 membered heterocyclyl, 5-10membered heteroaryl, (C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic)-,(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or heteroaryl-(C₁₋₁₂aliphatic)-;wherein any hydrogen atom is optionally and independently replaced byR¹³ and any set of two hydrogen atoms bound to the same atom isoptionally and independently replaced by carbonyl; R¹⁰ is halogen,—OR¹¹, —NO₂, —CN, —CF₃—OCF₃, —R¹¹, or —SR¹¹; wherein R¹¹ isC₁₋₄-aliphatic-; R¹¹ is C₁₋₄-aliphatic-; and R¹² is halogen, —OR¹¹,—NO₂—CN—CF₃—OCF₃, —R¹¹, or —SR⁹; R¹³ is —OR¹¹, —NO₂, —CN, —CF₃,—OCF₃—R¹¹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R¹¹)₂, —SR¹¹,—SOR¹¹, —SO₇R¹¹—SO₂N(R¹¹)₂—SO₃R¹¹, —C(O)R¹¹, —C(O)C(O)R¹¹,—C(O)C(O)OR¹¹, —C(O)C(O)N(R¹¹)₂, —C(O)CH₂C(O)R¹¹—C(S)R¹¹, —C(S)OR¹¹,—C(O)OR¹¹, —OC(O)R¹¹, —C(O)N(R¹¹)₂, —OC(O)N(R¹¹)₂, —C(S)N(R¹¹)₂,—(CH₂)₀₋₂NHC(O)R¹¹, —N(R¹¹)N(R¹¹)COR¹¹, —N(R¹¹)N(R¹¹)C(O)OR¹¹,—N(R¹¹)N(R¹¹)CON(R¹¹)₂, —N(R¹¹)SO₂R¹¹, —N(R¹¹)SO₂N(R¹¹)₂,—N(R¹¹)C(O)OR¹¹, —N(R¹¹)C(O)R¹¹, —N(R¹¹)C(S)R¹¹, —N(R¹¹)C(O)N(R¹¹)₂,—N(R¹¹)C(S)N(R¹¹)₂, —N(COR¹¹)COR¹¹, —N(OR¹¹)R¹¹, —C(═NH)N(R¹¹)₂,—C(O)N(OR¹¹)R¹¹, —C(═NOR¹¹)R¹¹, —OP(O)(OR¹¹)₂, —P(O)(R¹¹)₂,—P(O)(OR¹¹)₂, and —P(O)(H)(OR¹¹); R¹¹ is hydrogen, C₁₋₁₂ aliphatic,C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 membered heterocyclyl, 5-10membered heteroaryl, (C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic),(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or heteroaryl-(C₁₋₁₂aliphatic)-; orsingle stereoisomers, mixtures of stereoisomers, or pharmaceuticallyacceptable salts thereof; whereby the symptom of the HIV-1 infection inthe patient having the HIV-1 infection is ameliorated.
 22. The methodaccording to claim 21, wherein the compound has the following formula:


23. The method according to claim 22, wherein a stereoisomer of thecompound of Formula 19 is selected from the group consisting of:


24. The method according to claim 21, wherein the pharmaceuticalcomposition comprises a mixture of stereoisomers having the formula:


25. The method according to claim 21, wherein the method furthercomprises the step of: (b) administering to the patient highly activeantiretroviral therapy (HAART).
 26. The method according to claim 25,wherein steps (a) and (b) are performed by co-administration.
 27. Themethod according to claim 21, wherein the method further comprises thestep of: (b) administering to the patient an anti-HIV-1 compound. 28.The method according to claim 27 wherein steps (a) and (b) are performedby co-administration.
 29. The method according to claim 27, wherein theanti-HIV-1 compound is selected from the group consisting of anucleoside reverse transcriptase inhibitor, a non-nucleoside reversetranscriptase inhibitor, and a protease inhibitor.
 30. The methodaccording to claim 29, wherein the nucleoside reverse transcriptaseinhibitor is selected from the group consisting of ZIDOVUDINE® (AZT),LAMIVUDINE® (3TC), and STAVUDINE® (d4T).
 31. The method according toclaim 29, wherein the non-nucleoside reverse transcriptase inhibitor isNEVIRAPINE® (BI-RG-587).
 32. The method according to claim 29, whereinthe protease inhibitor is selected from the group consisting ofINDINAVIR® (MK-639), NELFINAVIR® (AG-1343), VIRACEPT®, SAQINAVIR®(Ro-31-8959), INVIRASE®, FORTOVASE®, RITONAVIR® (ABT-538), NORVIR®,indinavir (MK-639), CEIXIVAN®, amprenavir (141W94), AGENERASE®,02139-4211, LASINAVIR® (BMS-234475), CPGP-61755, DMP-450, an azapeptide,ABT-378, and AG-1549.
 33. The method according to claim 27, wherein theanti-HIV-1 compound is selected from the group consisting ofhydroxyurea, ribavirin, interleukin (IL)-2, PROLEUKIN®, IL-12,pentafuside (DP-178, T-20), and Yissum Project No.
 11607. 34. The methodaccording to claim 21, wherein step (a) comprises administering thepharmaceutical composition via a route selected from the groupconsisting of orally, rectally, vaginally, intradermal, subdermal,intravenously, intramuscularly, intranasal, intracerebral,intratracheal, intraarterial, intraperitoneal, intravesical,intrapleural, intracoronary or parenteral.
 35. The method according toclaim 21, wherein step (a) comprises administering the pharmaceuticalcomposition in form of a tablet or capsule.
 36. The method according toclaim 21, wherein step (a) comprises administering the pharmaceuticalcomposition in form of a liquid preparation.
 37. The method according toclaim 21, wherein the pharmaceutical composition further comprises acomponent selected from the group consisting of a preservative, anadjuvant, a stabilizing agent, a wetting agent, an emulsifying agent, asolution promoter, a salt for regulating osmotic pressure, a buffer, adiluent, a filler, a lubricant, a binder, a disintegrate, an absorbent,a colorant, a flavor compound and a sweetener.
 38. The method accordingto claim 21, wherein step (a) comprises administering the compound in anamount of between about 5 to 500 mg.
 39. The method according to claim21, wherein step (a) comprises administering the compound to the patientin a range selected from the group consisting of from about 0.1 mg/kg ofpatient weight to about 1 g/kg of patient weight, from about 5 mg/kg ofpatient weight to about 500 mg/kg of patient weight, from about 10 mg/kgof patient weight to about 250 mg/kg of patient weight, and from about25 mg/kg of patient weight to about 150 mg/kg of patient weight.
 40. Themethod according to claim 21, wherein step (a) comprises administeringthe compound in an amount of about 10 mg/kg of patient weight.
 41. Themethod according to claim 21, wherein step (a) comprises administeringthe compound to the patient for a period comprising at least threeconsecutive days, at least five consecutive days, at least 10consecutive days, at least 20 consecutive days, at least 30 consecutivedays or at least 40 consecutive days.
 42. The method according to claim21, wherein the symptom of the HIV-1 infection is a reduced CD4 T-cellcount and the method further comprising the step of: (b) measuring thepatient's CD4 T-cell count.
 43. The method according to claim 42,wherein the patient has a reduced T-cell count of less than 1,000/mm³.44. The method according to claim 21, wherein the symptom of the HIV-1infection is an increased interleukin (IL)-1β level and the methodfurther comprising the step of: (b) measuring the patient's IL-1β level.45. The method according to claim 21, wherein the patient has developeda resistance against an anti-HIV-1 compound.
 46. A method for preventingreduction of a CD4 T-cell count in a subject, the method comprising thestep of: (a) administering to a subject a pharmaceutical compositioncomprising a pharmaceutically effective amount of a compound having theformula:

wherein Y is

R¹ is H, C₁₋₁₂aliphatic, C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl,(C₃₋₁₀cycloalkyl)-(C₁₋₁₂aliphatic)-, cycloalkenyl-(C₁₋₁₂aliphatic)-,(C₆₋₁₀ aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or (5-10 memberedheteroaryl)-(C₁₋₁₂aliphatic)-, wherein any hydrogen atom is optionallyand independently replaced by R⁸ and any set of two hydrogen atoms boundto the same atom is optionally and independently replaced by carbonyl;Ring A is

wherein, in each ring, any hydrogen atom is optionally and independentlyreplaced by R⁴ and any set of two hydrogen atoms bound to the same atomis optionally and independently replaced by carbonyl; when Ring A is

then R is R³C(O)—, HC(O), R³SO₂—, R³OC(O), (R³)₂NC(O), (R³)(H)NC(O),R³C(O)C(O)—, R³—, (R³)₂NC(O)C(O), (R³)(H)NC(O)C(O), or R³OC(O)C(O)—; andR³ is C₁₋₁₂aliphatic, C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl,(C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic)-, (C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-,(5-10 membered heterocyclyl)-(C₁₋₁₂aliphatic)-, or (5-10 memberedheteroaryl)-(C₁₋₁₂aliphatic)-; or two R³ groups bound to the same atomform together with that atom a 3-10 membered aromatic or nonaromaticring; wherein any ring is optionally fused to an C₆₋₁₀aryl, 5-10membered heteroaryl, C₃₋₁₀cycloalkyl, or 5-10 membered heterocyclyl;wherein up to 3 aliphatic carbon atoms may be replaced by a groupselected from O, N, NR⁹, S, SO, and SO₂, wherein R³ is substituted withup to 6 substituents independently selected from R^(8′); when Ring A is

then R is R³C(O)—, as shown below:

and R³ is phenyl, thiophene, or pyridine, wherein each ring isoptionally substituted with up to 5 groups independently selected fromR^(8′), and wherein at least one position on the phenyl, thiophene, orpyridine adjacent to bond x is substituted by R¹², wherein R¹² has nomore than 5 straight-chained atoms; R⁴ is halogen, —OR⁹, —NO₂—CN—CF₃,—OCF₁, —R⁹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁹)₂, —SR⁹, —SOR⁹,—SO₂R⁹—SO₂N(R⁹)₂, —SO₃R⁹, —C(O)R⁹, —C(O)C(O)R⁹, —C(O)C(O)OR⁹,—C(O)C(O)N(R⁹)₂, —C(O)CH₂C(O)R⁹, —C(S)R⁹, —C(S)OR⁹, —C(O)OR⁹, —OC(O)R⁹,—C(O)N(R⁹)₂, —OC(O)N(R⁹)₂, —C(S)N(R⁹)₂, —(CH₂)₀₋₂ NHC(O)R⁹,—N(R⁹)N(R⁹)COR⁹, —N(R⁹)N(R⁹)C(O)OR⁹, —N(R⁹)N(R⁹)CON(R⁹)₂, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂N(R⁹)₂, —N(R⁹)C(O)OR⁹, —N(R⁹)C(O)R⁹, —N(R⁹)C(S)R⁹,—N(R⁹)C(O)N(R⁹)₂, —N(R⁹)C(S)N(R⁹)₂—N(COR⁹)COR⁹, —N(OR⁹)R⁹,—C(═NH)N(R⁹)₂, —C(O)N(OR⁹)R⁹, —C(═NOR⁹)R⁹, —OP(O)(OR⁹)₂, —P(O)(R⁹)₂,—P(O)(OR⁹)₂, or —P(O)(H)(OR⁹); R² is —C(R⁵)(R⁶)(R⁷), C₆₋₁₀aryl, 5-10membered heteroaryl, or C₃₋₇ cycloalkyl; R⁵ is H or a C₁₋₆straight-chained or branched alkyl; R⁶ is H or a C₁₋₆ straight-chainedor branched alkyl; R⁷ is —CF₃, —C₃₋₇cycloalkyl, C₆₋₁₀aryl, 5-10 memberedheteroaryl, heterocycle, or a C₁₋₆ straight-chained or branched alkyl,wherein each carbon atom of the alkyl is optionally and independentlysubstituted with R¹⁰; or R⁵ and R⁷ taken together with the carbon atomto which they are attached form a 3-10 membered cycloaliphatic; R⁸ andR^(8′) are each independently halogen, —OR⁹, —NO₂, —CN, —CF₃, —OCF₃,—R⁹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁹)₂, —SR⁹, —SOR⁹,—SO₂R⁹, —SO₂N(R⁹)₂—SO₃R⁹, —C(O)R⁹, —C(O)C(O)R⁹, —C(O)C(O)OR⁹,—C(O)C(O)N(R⁹)₂, —C(O)CH₂C(O)R⁹, —C(S)R⁹, —C(S)OR⁹, —C(O)OR⁹, —OC(O)R⁹,—C(O)N(R⁹)₂, —OC(O)N(R⁹)₂, —C(S)N(R⁹)₂, —(CH₂)₀₋₂NHC(O)R⁹,—N(R⁹)N(R⁹)COR⁹, —N(R⁹)N(R⁹)C(O)OR⁹, —N(R⁹)N(R⁹)CON(R⁹)₂, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂N(R⁹)₂, —N(R⁹)C(O)OR⁹, —N(R⁹)C(O)R⁹, —N(R⁹)C(S)R⁹,—N(R⁹)C(O)N(R⁹)₂, —N(R⁹)C(S)N(R⁹)₂, —N(COR⁹)COR⁹, —N(OR⁹)R⁹,—C(═NH)N(R⁹)₂, —C(O)N(OR⁹)R⁹, —C(═NOR⁹)R⁹, —OP(O)(OR⁹)₂, —P(O)(R⁹)₂,—P(O)(OR⁹)₂, and —P(O)(H)(OR⁹); R⁹ is hydrogen, C₁₋₁₂aliphatic,C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 membered heterocyclyl, 5-10membered heteroaryl, (C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic)-,(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or heteroaryl-(C₁₋₁₂aliphatic)-;wherein any hydrogen atom is optionally and independently replaced byR¹³ and any set of two hydrogen atoms bound to the same atom isoptionally and independently replaced by carbonyl; R¹⁰ is halogen,—OR¹¹, —NO₂, —CN, —CF₃—OCF₃, —R¹¹ or —SR¹¹; wherein R¹¹ isC₁₋₄-aliphatic-; R¹¹ is C₁₋₄-aliphatic-; and R¹² is halogen, —OR¹¹,—NO₂—CN—CF₃—OCF₃, —R¹¹, or —SR⁹; R¹³ is —OR¹¹, —NO₂, —CN, —CF₃, —OCF₃,—R¹¹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R¹¹)₂, —SR¹¹, —SOR¹¹,—SO₇R¹¹—SO₂N(R¹¹)₂—SO₃R¹¹, —C(O)R¹¹, —C(O)C(O)R¹¹, —C(O)C(O)OR¹¹,—C(O)C(O)N(R¹¹)₂, —C(O)CH₂C(O)R¹¹—C(S)R¹¹, —C(S)OR¹¹, —C(O)OR¹¹,—OC(O)R¹¹, —C(O)N(R¹¹)₂, —OC(O)N(R¹¹)₂, —C(S)N(R¹¹)₂,—(CH₂)₀₋₂NHC(O)R¹¹, —N(R¹¹)N(R¹¹)COR¹¹, —N(R¹¹)N(R¹¹)C(O)OR¹¹,—N(R¹¹)N(R¹¹)CON(R¹¹)₂, —N(R¹¹)SO₂R¹¹, —N(R¹¹)SO₂N(R¹¹)₂,—N(R¹¹)C(O)OR¹¹, —N(R¹¹)C(O)R¹¹, —N(R¹¹)C(S)R¹¹, —N(R¹¹)C(O)N(R¹¹)₂,—N(R¹¹)C(S)N(R¹¹)₂, —N(COR¹¹)COR¹¹, —N(OR¹¹)R¹¹, —C(═NH)N(R¹¹)₂,—C(O)N(OR¹¹)R¹¹, —C(═NOR¹¹)R¹¹, —OP(O)(OR¹¹)₂, —P(O)(R¹¹)₂,—P(O)(OR¹¹)₂, and —P(O)(H)(OR¹¹); R¹¹ is hydrogen, C₁₋₁₂aliphatic,C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 membered heterocyclyl, 5-10membered heteroaryl, (C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic),(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or heteroaryl-(C₁₋₁₂aliphatic)-; orsingle stereoisomers, mixtures of stereoisomers, or pharmaceuticallyacceptable salts thereof; whereby the CD4 T-cell count in the subjectafter performing step (a) is higher or not reduced compared to the CD4T-cell count in the subject before performing step (a).
 47. The methodaccording to claim 46, wherein the compound has the following formula:


48. The method according to claim 47, wherein a stereoisomer of thecompound of Formula 19 is selected from the group consisting of:


49. A method for prophylactic treatment of a subject, the methodcomprising the step of: (a) administering to a subject a pharmaceuticalcomposition comprising a pharmaceutically effective amount of a compoundhaving the formula:

wherein Y is

R¹ is H, C₁₋₁₂aliphatic, C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl,(C₃₋₁₀cycloalkyl)-(C₁₋₁₂aliphatic)-, cycloalkenyl-(C₁₋₁₂aliphatic)-,(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or (5-10 memberedheteroaryl)-(C₁₋₁₂aliphatic)-, wherein any hydrogen atom is optionallyand independently replaced by R⁸ and any set of two hydrogen atoms boundto the same atom is optionally and independently replaced by carbonyl;Ring A is

wherein, in each ring, any hydrogen atom is optionally and independentlyreplaced by R⁴ and any set of two hydrogen atoms bound to the same atomis optionally and independently replaced by carbonyl; when Ring A is

then R is R³C(O)—, HC(O), R³SO₂—, R³OC(O), (R³)₂NC(O), (R³)(H)NC(O),R³C(O)C(O)—, R³—, (R³)₂NC(O)C(O), (R³)(H)NC(O)C(O), or R³OC(O)C(O)—; andR³ is C₁₋₁₂aliphatic, C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 memberedheterocyclyl, 5-10 membered heteroaryl,(C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic)-, (C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-,(5-10 membered heterocyclyl)-(C₁₋₁₂aliphatic)-, or (5-10 memberedheteroaryl)-(C₁₋₁₂aliphatic)-; or two R³ groups bound to the same atomform together with that atom a 3-10 membered aromatic or nonaromaticring; wherein any ring is optionally fused to an C₆₋₁₀aryl, 5-10membered heteroaryl, C₃₋₁₀cycloalkyl, or 5-10 membered heterocyclyl;wherein up to 3 aliphatic carbon atoms may be replaced by a groupselected from O, N, NR⁹, S, SO, and SO₂, wherein R³ is substituted withup to 6 substituents independently selected from R^(8′); when Ring A is

then R is R³C(O)—, as shown below:

and R³ is phenyl, thiophene, or pyridine, wherein each ring isoptionally substituted with up to 5 groups independently selected fromR^(8′), and wherein at least one position on the phenyl, thiophene, orpyridine adjacent to bond x is substituted by R¹², wherein R¹² has nomore than 5 straight-chained atoms; R⁴ is halogen, —OR⁹, —NO₂—CN—CF₃,—OCF₁, —R⁹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁹)₂, —SR⁹, —SOR⁹,—SO₂R⁹—SO₂N(R⁹)₂, —SO₃R⁹, —C(O)R⁹, —C(O)C(O)R⁹, —C(O)C(O)OR⁹,—C(O)C(O)N(R⁹)₂, —C(O)CH₂C(O)R⁹, —C(S)R⁹, —C(S)OR⁹, —C(O)OR⁹, —OC(O)R⁹,—C(O)N(R⁹)₂, —OC(O)N(R⁹)₂, —C(S)N(R⁹)₂, —(CH₂)₀₋₂ NHC(O)R⁹,—N(R⁹)N(R⁹)COR⁹, —N(R⁹)N(R⁹)C(O)OR⁹, —N(R⁹)N(R⁹)CON(R⁹)₂, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂N(R⁹)₂, —N(R⁹)C(O)OR⁹, —N(R⁹)C(O)R⁹, —N(R⁹)C(S)R⁹,—N(R⁹)C(O)N(R⁹)₂, —N(R⁹)C(S)N(R⁹)₂—N(COR⁹)COR⁹, —N(OR⁹)R⁹,—C(═NH)N(R⁹)₂, —C(O)N(OR⁹)R⁹, —C(═NOR⁹)R⁹, —OP(O)(OR⁹)₂, —P(O)(R⁹)₂,—P(O)(OR⁹)₂, or —P(O)(H)(OR⁹); R² is —C(R⁵)(R⁶)(R⁷), C₆₋₁₀aryl, 5-10membered heteroaryl, or C₃₋₇ cycloalkyl; R⁵ is H or a C₁₋₆straight-chained or branched alkyl; R⁶ is H or a C₁₋₆ straight-chainedor branched alkyl; R⁷ is —CF₃, —C₃₋₇cycloalkyl, C₆₋₁₀aryl, 5-10 memberedheteroaryl, heterocycle, or a C₁₋₆ straight-chained or branched alkyl,wherein each carbon atom of the alkyl is optionally and independentlysubstituted with R¹⁰; or R⁵ and R⁷ taken together with the carbon atomto which they are attached form a 3-10 membered cycloaliphatic; R⁸ andR^(8′) are each independently halogen, —OR⁹, —NO₂, —CN, —CF₃, —OCF₃,—R⁹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R⁹)₂, —SR⁹, —SOR⁹,—SO₂R⁹, —SO₂N(R⁹)₂—SO₃R⁹, —C(O)R⁹, —C(O)C(O)R⁹, —C(O)C(O)OR⁹,—C(O)C(O)N(R⁹)₂, —C(O)CH₂C(O)R⁹, —C(S)R⁹, —C(S)OR⁹, —C(O)OR⁹, —OC(O)R⁹,—C(O)N(R⁹)₂, —OC(O)N(R⁹)₂, —C(S)N(R⁹)₂, —(CH₂)₀₋₂NHC(O)R⁹,—N(R⁹)N(R⁹)COR⁹, —N(R⁹)N(R⁹)C(O)OR⁹, —N(R⁹)N(R⁹)CON(R⁹)₂, —N(R⁹)SO₂R⁹,—N(R⁹)SO₂N(R⁹)₂, —N(R⁹)C(O)OR⁹, —N(R⁹)C(O)R⁹, —N(R⁹)C(S)R⁹,—N(R⁹)C(O)N(R⁹)₂, —N(R⁹)C(S)N(R⁹)₂, —N(COR⁹)COR⁹, —N(OR⁹)R⁹,—C(═NH)N(R⁹)₂, —C(O)N(OR⁹)R⁹, —C(═NOR⁹)R⁹, —OP(O)(OR⁹)₂, —P(O)(R⁹)₂,—P(O)(OR⁹)₂, and —P(O)(H)(OR⁹); R⁹ is hydrogen, C₁₋₁₂aliphatic,C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 membered heterocyclyl, 5-10membered heteroaryl, (C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic)-,(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or heteroaryl-(C₁₋₁₂aliphatic)-;wherein any hydrogen atom is optionally and independently replaced byR¹³ and any set of two hydrogen atoms bound to the same atom isoptionally and independently replaced by carbonyl; R¹⁰ is halogen,—OR¹¹, —NO₂, —CN, —CF₃—OCF₃, —R¹¹, or —SR¹¹; wherein R¹¹ isC₁₋₄-aliphatic-; R¹¹ is C₁₋₄-aliphatic-; and R¹² is halogen, —OR¹¹,—NO₂—CN—CF₃—OCF₃, —R¹¹, or —SR⁹; R¹³ is —OR¹¹, —NO₂, —CN, —CF₃, —OCF₃,—R¹¹, 1,2-methylenedioxy, 1,2-ethylenedioxy, —N(R¹¹)₂, —SR¹¹, —SOR¹¹,—SO₇R¹¹—SO₂N(R¹¹)₂—SO₃R¹¹, —C(O)R¹¹, —C(O)C(O)R¹¹, —C(O)C(O)OR¹¹,—C(O)C(O)N(R¹¹)₂, —C(O)CH₂C(O)R¹¹—C(S)R¹¹, —C(S)OR¹¹, —C(O)OR¹¹,—OC(O)R¹¹, —C(O)N(R¹¹)₂, —OC(O)N(R¹¹)₂, —C(S)N(R¹¹)₂,—(CH₂)₀₋₂NHC(O)R¹¹, —N(R¹¹)N(R¹¹)COR¹¹, —N(R¹¹)N(R¹¹)C(O)OR¹¹,—N(R¹¹)N(R¹¹)CON(R¹¹)₂, —N(R¹¹)SO₂R¹¹, —N(R¹¹)SO₂N(R¹¹)₂,—N(R¹¹)C(O)OR¹¹, —N(R¹¹)C(O)R¹¹, —N(R¹¹)C(S)R¹¹, —N(R¹¹)C(O)N(R¹¹)₂,—N(R¹¹)C(S)N(R¹¹)₂, —N(COR¹¹)COR¹¹, —N(OR¹¹)R¹¹, —C(═NH)N(R¹¹)₂,—C(O)N(OR¹¹)R¹¹, —C(═NOR¹¹)R¹¹, —OP(O)(OR¹¹)₂, —P(O)(R¹¹)₂,—P(O)(OR¹¹)₂, and —P(O)(H)(OR¹¹); R¹¹ is hydrogen, C₁₋₁₂ aliphatic,C₃₋₁₀cycloaliphatic, C₆₋₁₀aryl, 5-10 membered heterocyclyl, 5-10membered heteroaryl, (C₃₋₁₀cycloaliphatic)-(C₁₋₁₂aliphatic),(C₆₋₁₀aryl)-(C₁₋₁₂aliphatic)-, (5-10 memberedheterocyclyl)-(C₁₋₁₂aliphatic)-, or heteroaryl-(C₁₋₁₂aliphatic)-; orsingle stereoisomers, mixtures of stereoisomers, or pharmaceuticallyacceptable salts thereof; whereby the prophylactic treatment reducesdevelopment of a clinical symptom of a pathological condition associatedwith or developing in a subject as a consequence of HIV-1 infection orAIDS.
 50. The method according to claim 49, wherein the compound has thefollowing formula: